Hybrid tomato plant named HM 5235

ABSTRACT

A novel hybrid tomato plant, designated HM5235 is disclosed. The invention relates to the seeds of tomato hybrid HM5235, to the plants and plant parts of hybrid tomato HM5235, and to methods for producing a tomato plant by crossing the hybrid tomato HM5235 with itself or another tomato plant. The invention further relates to methods for producing a tomato plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other tomato plants derived from the hybrid tomato plant HM5235.

FIELD OF THE INVENTION

The present invention relates to the field of agriculture, to new anddistinctive hybrid tomato plants, such as hybrid plants designatedHM5235 and to methods of making and using such hybrids.

BACKGROUND OF THE INVENTION

The following description includes information that may be useful inunderstanding the present invention. It is not an admission that any ofthe information provided herein is prior art or relevant to thepresently claimed inventions, or that any publication specifically orimplicitly referenced is prior art.

Tomato is an important and valuable vegetable crop. Thus, a continuinggoal of plant breeders is to develop stable, high yielding tomatohybrids that are agronomically sound or unique. The reasons for thisgoal are to maximize the amount of fruit produced on the land used(yield) as well as to improve the fruit appearance, the fruit shape andsize, eating and processing qualities and/or the plant agronomic andhorticultural qualities. To accomplish this goal, the tomato breedermust select and develop tomato plants that have the traits that resultin superior parental lines that combine to produce superior hybrids.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope.

In various embodiments, one or more of the above-described problems havebeen reduced or eliminated, while other embodiments are directed toother improvements.

According to the invention, in some embodiments there is provided anovel hybrid tomato, designated HM5235.

This invention thus relates to the seeds of hybrid tomato designatedHM5235, to the plants or parts thereof of hybrid tomato designatedHM5235, to plants or parts thereof consisting essentially of thephenotypic and morphological characteristics of hybrid tomato designatedHM5235, and/or having all the physiological and morphologicalcharacteristics of hybrid tomato designated HM5235, and/or having one ormore or all of the characteristics of hybrid tomato designated HM5235listed in Table 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions,and/or having one or more of the physiological and morphologicalcharacteristics of hybrid tomato designated HM5235 listed in Table 1including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions and/or having all thephysiological and morphological characteristics of hybrid tomatodesignated HM5235 listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions and/or having all the physiological andmorphological characteristics of hybrid tomato designated HM5235 listedin Table 1 when grown in the same environmental conditions. Theinvention also relates to variants, mutants and trivial modifications ofthe seed or plant of hybrid tomato designated HM5235.

Plant parts of the hybrid tomato plant of the present invention are alsoprovided, such as, a scion, a rootstock, a fruit, leaf, flower,peduncle, stalk, root, anther cell, pollen or ovule obtained from thehybrid plant. The present invention provides fruit of the hybrid tomatoof the present invention. Such fruit and parts thereof could be used asfresh products for consumption or in processes resulting in processedproducts such as food products comprising one or more harvested part ofthe hybrid tomato designated HM5235, such as prepared fruit or partsthereof, canned fruit or parts thereof, freeze dried or frozen fruit orparts thereof, diced fruits, juice, prepared fruit cuts, cannedtomatoes, pastes, sauces, puree, catsups and the like. All such productsare part of the present invention and the like. The harvested part orfood product can be or can comprise hybrid tomato fruit from hybridtomato designated HM5235. The food products might have undergone one ormore processing steps such as, but not limited to cutting, washing,mixing, frizzing, canning, etc. All such products are part of thepresent invention.

The plants and seeds of the present invention include those that may beof an essentially derived variety as defined in section 41(3) of thePlant Variety Protection Act of The United States of America, e.g., avariety that is predominantly derived from hybrid tomato designatedHM5235 or from a variety that i) is predominantly derived from hybridtomato designated HM5235, while retaining the expression of theessential characteristics that result from the genotype or combinationof genotypes of hybrid tomato designated HM5235; ii) is clearlydistinguishable from hybrid tomato designated HM5235; and iii) exceptfor differences that result from the act of derivation, conforms to theinitial variety in the expression of the essential characteristics thatresult from the genotype or combination of genotypes of the initialvariety.

In another aspect, the present invention provides regenerable cells. Insome embodiments, the regenerable cells are for use in tissue culture ofhybrid tomato designated HM5235. In some embodiments, the tissue cultureis capable of regenerating plants consisting essentially of thephenotypic and morphological characteristics of hybrid tomato designatedHM5235, and/or having all the phenotypic and morphologicalcharacteristics of hybrid tomato designated HM5235, and/or having thephysiological and morphological characteristics of hybrid tomatodesignated HM5235, and/or having the characteristics of hybrid tomatodesignated HM5235. In one embodiment, the regenerated plants have thecharacteristics of pep hybrid tomato designated HM5235 listed in Tables1 including but not limited to as determined at the 5% significancelevel when grown in the same environmental conditions.

In some embodiments, the plant parts and cells used to produce suchtissue cultures will be embryos, meristematic cells, seeds, callus,pollen, leaves, anthers, pistils, roots, root tips, stems, petioles,fruits, cotyledons, hypocotyls, ovaries, seed coat, fruits, stalks,endosperm, flowers, axillary buds or the like. Protoplasts produced fromsuch tissue culture are also included in the present invention. Thetomato shoots, roots and whole plants regenerated from the tissueculture, as well as the fruit produced by said regenerated plants arealso part of the invention. In some embodiments, the whole plantsregenerated from the tissue culture have one, more than one, or all ofthe physiological and morphological characteristics of tomato hybriddesignated HM5235 listed in Table 1 including but not limited to asdetermined at the 5% significance level when grown in the sameenvironmental conditions.

The invention also discloses methods for vegetatively propagating aplant of the present invention. In the present application, vegetativelypropagating can be interchangeably used with vegetative reproduction. Insome embodiments, the methods comprise collecting a part of a hybridtomato designated HM5235 and regenerating a plant from said part. Insome embodiments, the part can be for example a stem cutting that isrooted into an appropriate medium according to techniques known by theone skilled in the art. Plants, plant parts and fruits thereof producedby such methods are also included in the present invention. In anotheraspect, the plants and fruits thereof produced by such methods consistessentially of the phenotypic and morphological characteristics ofhybrid tomato designated HM5235, and/or having all the phenotypic andmorphological characteristics of hybrid tomato designated HM5235 and/orhaving the physiological and morphological characteristics of hybridtomato designated HM5235 and/or having the characteristics of hybridtomato designated HM5235. In some embodiments, plants produced by suchmethods consist of one, more than one, or all phenotypic andmorphological characteristics of tomato hybrid designated listed inTable 1 including but not limited to as determined at the 5%significance level when grown in the same environmental conditions.

Further included in the invention are methods for producing fruitsand/or seeds from the hybrid tomato designated HM5235. In someembodiments, the methods comprise growing a hybrid tomato designatedHM5235 to produce tomato fruits and/or seeds. In some embodiments, themethods further comprise harvesting the hybrid tomato fruits and/orseeds. Such fruits and/or seeds are part of the present invention.

Also included in this invention are methods for producing a tomatoplant. In some embodiments, the tomato plant is produced by crossing thehybrid tomato designated HM5235 with itself or another tomato plant. Insome embodiments, the other plant can be a tomato hybrid or line. Whencrossed with an inbred line, in some embodiments, a “three-way cross” isproduced. When crossed with itself or with another, different hybridtomato, in some embodiments, a “four-way” cross is produced. Such threeand four-way hybrid seeds and plants produced by growing said three andfour-way hybrid seeds are included in the present invention. Methods forproducing a three and four-way hybrid tomato seed comprising crossinghybrid tomato designated HM5235 tomato plant with a different tomatoline or hybrid and harvesting the resultant hybrid tomato seed are alsopart of the invention. The hybrid tomato seeds produced by the methodcomprising crossing hybrid tomato designated HM5235 tomato plant with adifferent tomato plant and harvesting the resultant hybrid tomato seedare included in the invention, as are included the hybrid tomato plantor parts thereof and seeds produced by said grown hybrid tomato plants.

Further included in the invention are methods for producing tomato seedsand plants made thereof. In some embodiments, the methods compriseself-pollinating the hybrid tomato designated HM5235 and harvesting theresultant hybrid seeds. Tomato seeds produced by such method are alsopart of the invention.

In another embodiment, this invention relates to methods for producing ahybrid tomato designated HM5235 from a collection of seeds. In someembodiments, the collection contains both seeds of inbred parent line(s)of hybrid tomato designated HM5235 seeds and hybrid seeds of HM5235.Such a collection of seeds might be a commercial bag of seeds. In someembodiments, said methods comprise planting the collection of seeds.When planted, the collection of seeds will produce inbred parent linesof hybrid tomato HM5235 and hybrid plants from the hybrid seeds ofHM5235. In some embodiments, said inbred parent lines of hybrid tomatodesignated HM5235 plants are identified as having a decreased vigorcompared to the other plants (i.e. hybrid plants) grown from thecollection of seeds. In some embodiments, said decreased vigor is due tothe inbreeding depression effect and can be identified for example by aless vigorous appearance for vegetative and/or reproductivecharacteristics including a shorter plant height, small fruit size,fruit shape, fruit color or other characteristics. In some embodiments,seeds of the inbred parent lines of the hybrid tomato HM5235 arecollected and, if new inbred plants thereof are grown and crossed in acontrolled manner with each other, the hybrid tomato HM5235 will berecreated.

This invention also relates to methods for producing other tomato plantsderived from hybrid tomato HM5235 and to the tomato plants derived bythe use of those methods.

In some embodiments, such methods for producing a tomato plant derivedfrom the hybrid variety HM5235 comprise (a) self-pollinating the hybridtomato HM5235 plant at least once to produce a progeny plant derivedfrom tomato hybrid HM5235; In some embodiments, the methods furthercomprise (b) crossing the progeny plant derived from tomato hybridHM5235 with itself or a second tomato plant to produce a seed of aprogeny plant of a subsequent generation; In some embodiments, themethods further comprise (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the methods further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond tomato plant to produce a tomato plant further derived from thehybrid tomato HM5235. In further embodiments, steps (b), (c) and/or (d)are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or more generations toproduce a tomato plant derived from the hybrid tomato variety HM5235. Insome embodiments, within each crossing cycle, the second plant is thesame plant as the second plant in the last crossing cycle. In someembodiments, within each crossing cycle, the second plant is differentfrom the second plant in the last crossing cycle.

Another method for producing a tomato plant derived from the hybridvariety HM5235, comprises the steps of: (a) crossing the hybrid tomatoHM5235 plant with a second tomato plant to produce a progeny plantderived from tomato hybrid HM5235; In some embodiments, the methodfurther comprise (b) crossing the progeny plant derived from tomatohybrid HM5235 with itself or a second tomato plant to produce a seed ofa progeny plant of a subsequent generation; In some embodiments, themethod further comprise (c) growing the progeny plant of the subsequentgeneration; In some embodiments, the method further comprise (d)crossing the progeny plant of the subsequent generation with itself or asecond tomato plant to produce a tomato plant derived from the hybridtomato variety HM5235. In a further embodiment, steps (b), (c) and/or(d) are repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, or moregenerations to produce a tomato plant derived from the hybrid tomatovariety HM5235. In some embodiments, within each crossing cycle, thesecond plant is the same plant as the second plant in the last crossingcycle. In some embodiments, within each crossing cycle, the second plantis different from the second plant in the last crossing cycle.

In another aspect, the present invention provides methods of introducingor modifying one or more desired trait(s) into the hybrid tomato HM5235and plants or seeds obtained from such methods. The desired trait(s) maybe, but not exclusively, a single gene. In some embodiments, the gene isa dominant allele. In some embodiments, the gene is a partially dominantallele. In some embodiments, the gene is a recessive allele. In someembodiments, the gene or genes will confer such traits, including butnot limited to male sterility, herbicide resistance, insect resistance,resistance for bacterial, fungal, mycoplasma or viral disease, enhancedplant quality such as improved drought or salt tolerance, water-stresstolerance, improved standability, enhanced plant vigor, improved shelflife, delayed senescence or controlled ripening, enhanced nutritionalquality such as increased sugar content or increased sweetness,increased texture, flavor and aroma, improved fruit length and/or size,protection for color, fruit shape, uniformity, length or diameter,refinement or depth, lodging resistance, yield and recovery, improvefresh cut application, specific aromatic compounds, specific volatiles,flesh texture, specific nutritional components. For the presentinvention and the skilled artisan, disease is understood to include, butnot limited to fungal diseases, viral diseases, bacterial diseases,mycoplasm diseases, or other plant pathogenic diseases and a diseaseresistant plant will encompass a plant resistant to fungal, viral,bacterial, mycoplasm, and other plant pathogens. The gene or genes maybe naturally occurring tomato gene(s), mutant(s), or genes modifiedthrough the use of New Breeding Techniques. In some embodiments, themethod for introducing the desired trait(s) is a backcrossing processmaking use of a series of backcrosses to at least one of the parentlines of hybrid tomato HM5235 during which the desired trait(s) ismaintained by selection. The single gene conversion plants that can beobtained by the methods are included in the present invention.

When dealing with a gene that has been modified, for example through NewBreeding Techniques, the trait (genetic modification) could be directlymodified into the newly developed line/cultivar such as at least one ofthe parent lines of hybrid tomato HM5235. Alternatively, if the trait isnot modified into each newly developed line/cultivar such as at leastone of the parent lines of hybrid tomato HM5235, another typical methodused by breeders of ordinary skill in the art to incorporate themodified gene is to take a line already carrying the modified gene andto use such line as a donor line to transfer the modified gene into oneor more of the parents of the newly developed hybrid.

The same would apply for a naturally occurring trait or one arising fromspontaneous or induced mutations.

In some embodiments, the backcross breeding process of hybrid tomatoHM5235 comprises (a) crossing one of the parental inbred line plants ofHM5235 with plants of another line that comprise the desired trait(s) toproduce F1 progeny plants In some embodiments, the process furthercomprises (b) selecting the F1 progeny plants that have the desiredtrait(s) In some embodiments, the process further comprises (c) crossingthe selected F1 progeny plants with the parental inbred tomato lines ofhybrid HM5235 plants to produce backcross progeny plants In someembodiments, the process further comprises (d) selecting for backcrossprogeny plants that have the desired trait(s) and physiological andmorphological characteristics of the tomato parental inbred line ofhybrid tomato HM5235 to produce selected backcross progeny plants; Insome embodiments, the process further comprises (e) repeating steps (c)and (d) one, two, three, four, five six, seven, eight, nine or moretimes in succession to produce selected, second, third, fourth, fifth,sixth, seventh, eighth, ninth or higher backcross progeny plants thathave the desired trait(s) and otherwise consist essentially of thephenotypic and morphological characteristics of the parental inbredtomato line of hybrid tomato HM5235, and/or have the desired trait(s)and otherwise the phenotypic and morphological characteristics of theparental tomato inbred line of hybrid tomato HM5235, and/or have all thedesired trait(s) and otherwise the physiological and morphologicalcharacteristics of the parental inbred tomato line of tomato hybridHM5235 as determined in Table 1, including but not limited to when grownin the same environmental conditions or including but not limited to ata 5% significance level when grown in the same environmental conditions.The tomato plants or seed produced by the methods are also part of theinvention, as are the hybrid tomato HM5235 plants that comprised thedesired trait. Backcrossing breeding methods, well known to one skilledin the art of plant breeding will be further developed in subsequentparts of the specification.

In an embodiment of this invention is a method of making a backcrossconversion of hybrid tomato HM5235. In some embodiments, the methodcomprises crossing one of the parental tomato inbred line plants ofhybrid HM5235 with a donor plant comprising a mutant gene(s), anaturally occurring gene(s) or a gene(s) and/or sequences modifiedthrough New Breeding Techniques conferring one or more desired trait toproduce F1 progeny plants. In some embodiments, the method furthercomprises selecting an F1 progeny plant comprising the naturallyoccurring gene(s), mutant gene(s) or modified gene(s) and/or sequencesconferring the one or more desired trait; In some embodiments, themethod further comprises backcrossing the selected progeny plant to theparental tomato inbred line plants of hybrid HM5235. This method mayfurther comprise the step of obtaining a molecular marker profile of theparental tomato inbred line plants of hybrid HM5235 and using themolecular marker profile to select for the progeny plant with thedesired trait and the molecular marker profile of the parental tomatoinbred line plants of hybrid HM5235. In some embodiments, this methodfurther comprises crossing the backcross progeny plant HM5235 containingthe naturally occurring gene(s), the mutant gene(s) or the modifiedgene(s) and or sequences conferring the one or more desired trait withthe second parental inbred tomato line plants of hybrid tomato HM5235 inorder to produce the hybrid tomato HM5235 comprising the naturallyoccurring gene(s), the mutant gene(s) or modified gene(s) and/orsequences conferring the one or more desired traits. The plants or partsthereof produced by such methods are also part of the present invention.

In some embodiments of the invention, the number of loci that may bebackcrossed into the parental tomato inbred line of hybrid HM5235 is atleast 1, 2, 3, 4, 5, or more.

A single locus may contain several genes. A single locus conversion alsoallows for making one or more site specific changes to the plant genome,such as, without limitation, one or more nucleotide change, deletion,insertions, etc. In some embodiments, the single locus conversion isperformed by genome editing, a.k.a. genome editing with engineerednucleases (GEEN). In some embodiments, the genome editing comprisesusing one or more engineered nucleases. In some embodiments, theengineered nucleases include, but are not limited to Zinc fingernucleases (ZFNs), Transcription Activator-Like Effector Nucleases(TALENs), the CRISPR/Cas system, and engineered meganucleasere-engineered homing endonucleases and endonucleases for DNA guidedgenome editing (Gao et al., Nature Biotechnology (2016), doi:10.1038/nbt.3547). In some embodiments, the single locus conversionchanges one or several nucleotides of the plant genome. Such genomeediting techniques are some of the techniques now known by the personskilled in the art and herein are collectively referred to as “NewBreeding Techniques”.

The invention further provides methods for developing tomato plants in atomato plant breeding program using plant breeding techniques includingbut not limited to, recurrent selection, backcrossing, pedigreebreeding, genomic selection, molecular marker (Isozyme Electrophoresis,Restriction Fragment Length Polymorphisms (RFLPs), Randomly AmplifiedPolymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction(AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence CharacterizedAmplified Regions (SCARs), Amplified Fragment Length Polymorphisms(AFLPs), and Simple Sequence Repeats (SSRs) which are also referred toas Microsatellites, Single Nucleotide Polymorphism (SNP), etc.) enhancedselection, genetic marker enhanced selection and transformation. Seeds,tomato plants, and parts thereof produced by such breeding methods arealso part of the invention.

The invention also relates to variants, mutants and trivialmodifications of the seed or plant of the tomato hybrid HM5235 or inbredparental lines thereof. Variants, mutants and trivial modifications ofthe seed or plant of hybrid tomato HM5235 or inbred parental linesthereof can be generated by methods available to one skilled in the art,including but not limited to, mutagenesis (e.g., chemical mutagenesis,radiation mutagenesis, transposon mutagenesis, insertional mutagenesis,signature tagged mutagenesis, site-directed mutagenesis, and naturalmutagenesis), knock-outs/knock-ins, antisense and RNA interference andother techniques such as the New Breeding Techniques. For moreinformation of mutagenesis in plants, such as agents or protocols, seeAcquaah et al. (Principles of plant genetics and breeding,Wiley-Blackwell, 2007, ISBN 1405136464, 9781405136464, which is hereinincorporated by reference in its entity).

The invention also relates to a mutagenized population of the hybridtomato HM5235 and methods of using such populations. In someembodiments, the mutagenized population can be used in screening for newtomato plants which comprise one or more or all of the morphological andphysiological characteristics of hybrid tomato HM5235. In someembodiments, the new tomato plants obtained from the screening processcomprise all of the morphological and physiological characteristics ofthe tomato hybrid HM5235 and one or more additional or differentmorphological and physiological characteristics that the tomato hybridHM5235 does not have.

This invention also is directed to methods for producing a tomato plantby crossing a first parent tomato plant with a second parent tomatoplant wherein either the first or second parent tomato plant is a hybridtomato plant of HM5235. Further, both first and second parent tomatoplants can come from the hybrid tomato plant HM5235. Further, the hybridtomato plant HM5235 can be self-pollinated i.e. the pollen of a hybridtomato plant HM5235 can pollinate the ovule of the same hybrid tomatoplant HM5235. When crossed with another tomato plant, a hybrid seed isproduced. Such methods of hybridization and self-pollination are wellknown to those skilled in the art of breeding.

An inbred tomato line such as one of the parental lines of hybrid tomatoHM5235 has been produced through several cycles of self-pollination andis therefore to be considered as a homozygous line. An inbred line canalso be produced though the dihaploid system which involves doubling thechromosomes from a haploid plant or embryo thus resulting in an inbredline that is genetically stable (homozygous) and can be reproducedwithout altering the inbred line. Haploid plants could be obtained fromhaploid embryos that might be produced from microspores, pollen, anthercultures or ovary cultures or spontaneous haploidy. The haploid embryosmay then be doubled by chemical treatments such as by colchicine or bedoubled autonomously. The haploid embryos may also be grown into haploidplants and treated to induce the chromosome doubling. In either case,fertile homozygous plants are obtained. A hybrid variety is classicallycreated through the fertilization of an ovule from an inbred parentalline by the pollen of another, different inbred parental line. Due tothe homozygous state of the inbred line, the produced gametes carry acopy of each parental chromosome. As both the ovule and the pollen bringa copy of the arrangement and organization of the genes present in theparental lines, the genome of each parental line is present in theresulting F1 hybrid, theoretically in the arrangement and organizationcreated by the plant breeder in the original parental line.

As long as the homozygosity of the parental lines is maintained, theresulting hybrid cross shall be stable. The F1 hybrid is then acombination of phenotypic characteristics issued from two arrangementand organization of genes, both created by a person skilled in the artthrough the breeding process.

Still further, this invention also is directed to methods for producinga tomato plant derived from hybrid tomato HM5235 by crossing hybridtomato plant HM5235 with a second tomato plant. In some embodiments, themethods further comprise obtaining a progeny seed from the cross. Insome embodiments, the methods further comprise growing the progeny seed,and possibly repeating the crossing and growing steps with the tomatohybrid plant HM5235-derived plant from 0 to 7 or more times. Thus, anysuch methods using the hybrid tomato plant HM5235 are part of thisinvention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using hybrid tomato plantHM5235 as a parent are within the scope of this invention, includingplants derived from hybrid tomato plant HM5235. In some embodiments,such plants have one, more than one or all phenotypic and morphologicalcharacteristics of the tomato hybrid plant HM5235 listed in Table 1including but not limited to as determined at the 5% significance levelwhen grown in the same environmental conditions. In some embodiments,such plants might exhibit additional and desired characteristics ortraits such as high seed yield, high seed germination, seedling vigor,early maturity, high fruit yield, ease of fruit setting, diseasetolerance or resistance, lodging resistance and adaptability for soiland climate conditions. Consumer-driven traits, such as a preference fora given fruit size, fruit shape, fruit color, fruit texture, fruittaste, fruit firmness, fruit sugar content are other traits that may beincorporated into new tomato plants developed by this invention.

A tomato plant can also be propagated vegetatively. A part of the plant,for example a shoot tissue, is collected, and a new plant is obtainedfrom the part. Such part typically comprises an apical meristem of theplant. The collected part is transferred to a medium allowingdevelopment of a plantlet, including for example rooting or developmentof shoots, or is grafted onto a tomato plant or a rootstock prepared tosupport growth of shoot tissue. This is achieved using methods wellknown in the art. Accordingly, in one embodiment, a method ofvegetatively propagating a plant of the present invention comprisescollecting a part of a plant according to the present invention, e.g. ashoot tissue, and obtaining a plantlet from said part. In oneembodiment, a method of vegetatively propagating a plant of the presentinvention comprises: a) collecting tissue of a plant of the presentinvention; b) rooting said proliferated shoots to obtain rootedplantlets. In one embodiment, a method of vegetatively propagating aplant of the present invention comprises: a) collecting tissue of aplant of the present invention; b) cultivating said tissue to obtainproliferated shoots; c) rooting said proliferated shoots to obtainrooted plantlets. In one embodiment, such method further comprisesgrowing a plant from said plantlets. In one embodiment, a fruit isharvested from said plant. In one embodiment, the fruit is processedinto products such as canned tomato fruits and/or parts thereof, juice,freeze dried or frozen fruit and/or parts thereof, fresh or preparedfruit and/or parts thereof or pastes, sauces, puree, catsups and thelike.

The invention is also directed to the use of the hybrid tomato plantHM5235 in a grafting process. In one embodiment, the hybrid tomato plantHM5235 is used as the scion while in another embodiment, the hybridtomato plant HM5235 is used as a rootstock.

In some embodiments, the present invention teaches a seed of hybridtomato designated HM5235, wherein a representative sample of seed ofsaid hybrid is deposited under NCIMB No. 42747.

In some embodiments, the present invention teaches a tomato plant, or apart thereof, produced by growing the deposited HM5235 seed.

In some embodiments, the present invention teaches tomato plant parts,wherein the tomato part is selected from the group consisting of: aleaf, a flower, a fruit, a seed, an ovule, pollen, a cell, a rootstock,and a scion.

In some embodiments, the present invention teaches a tomato plant, or apart thereof, having all of the characteristics of hybrid HM5235 aslisted in Table 1 of this application including but not limited to whengrown in the same environmental conditions.

In some embodiments, the present invention teaches a tomato plant, or apart thereof, having all of the physiological and morphologicalcharacteristics of hybrid HM5235, wherein a representative sample ofseed of said hybrid was deposited under NCIMB No. 42747.

In some embodiments, the present invention teaches a tissue culture ofregenerable cells produced from the plant or plant part grown from thedeposited HM5235 seed, wherein cells of the tissue culture are producedfrom a plant part selected from the group consisting of protoplasts,embryos, meristematic cells, callus, pollen, ovules, flowers, seeds,leaves, roots, root tips, anthers, stems, petioles, fruits, axillarybuds, cotyledons and hypocotyls. In some embodiments, the plant partincludes protoplasts produced from a plant grown from the depositedHM5235 seed.

In some embodiments, the present invention teaches a compositioncomprising regenerable cells produced from the plant or plant part grownfrom the deposited hybrid HM5235 seed, or other plant part or plantcell. In some embodiments, the composition comprises a growth media. Insome embodiments, the growth media is solid or a synthetic cultivationmedium. In some embodiments, the composition is a tomato plantregenerated from the tissue culture from a plant grown from thedeposited HM5235 seed, said plant having the characteristics of hybridHM5235, wherein a representative sample of seed of said hybrid isdeposited under NCIMB No. 42747.

In some embodiments, the present invention teaches a tomato fruitproduced from the plant grown from the deposited HM5235 seed.

In some embodiments, methods of producing said tomato fruit comprise a)growing the tomato plant from deposited HM5235 seed to produce a tomatofruit, and b) harvesting said tomato fruit. In some embodiments, thepresent invention also teaches a tomato fruit produced by the method ofproducing tomato fruit and/or seed as described above.

In some embodiments, the present invention teaches methods for producinga tomato seed comprising crossing a first parent tomato plant with asecond parent tomato plant and harvesting the resultant tomato seed,wherein said first parent tomato plant and/or second parent tomato plantis the tomato plant produced from the deposited HM5235 seed or a tomatoplant having all of the characteristics of tomato hybrid HM5235 aslisted in Table 1 including but not limited to when grown in the sameenvironmental conditions.

In some embodiments, the present invention teaches methods for producinga tomato seed comprising self-pollinating the tomato plant grown fromthe deposited HM5235 seed and harvesting the resultant tomato seed.

In some embodiments, the present invention teaches the seed produced byany of the above described methods.

In some embodiments, the present invention teaches methods ofvegetatively propagating the tomato plant grown from the depositedHM5235 seed, said method comprising a) collecting part of a plant grownfrom the deposited HM5235 seed and b) regenerating a plant from saidpart.

In some embodiments, the method further comprises harvesting a fruitand/or seed from said vegetatively propagated plant.

In some embodiments, the present invention teaches the plant and thefruit and/or seed of plants vegetatively propagated from plant parts ofplants grown from the deposited HM5235 seed.

In some embodiments, the present invention teaches methods of producinga tomato plant derived from the hybrid variety HM5235. In someembodiment the methods comprise (a) self-pollinating the plant grownfrom the deposited HM5235 seed at least once to produce a progeny plantderived from tomato hybrid HM5235. In some embodiments, the methodfurther comprises (b) crossing the progeny plant derived from tomatohybrid HM5235 with itself or a second tomato plant to produce a seed ofa progeny plant of a subsequent generation; and; (c) growing the progenyplant of the subsequent generation from the seed, and crossing theprogeny plant of the subsequent generation with itself or a secondtomato plant to produce a tomato plant derived from the hybrid tomatovariety HM5235. In some embodiments said methods further comprise thestep of: (d) repeating steps b) and/or c) for at least 1, 2, 3, 4, 5, 6,7, or more generation to produce a tomato plant derived from the hybridtomato variety HM5235.

In some embodiments, the present invention teaches methods of producinga tomato plant derived from the hybrid variety HM5235, the methodscomprising (a) crossing the plant grown from the deposited HM5235 seedwith a second tomato plant to produce a progeny plant derived fromtomato hybrid HM5235. In some embodiments, the method further comprises;(b) crossing the progeny plant derived from tomato hybrid HM5235 withitself or a second tomato plant to produce a seed of a progeny plant ofa subsequent generation; and; (c) growing the progeny plant of thesubsequent generation from the seed; (d) crossing the progeny plant ofthe subsequent generation with itself or a second tomato plant toproduce a tomato plant derived from the hybrid tomato variety HM5235. Insome embodiments said methods further comprise the steps of: (e)repeating step (b), (c) and/or (d) for at least 1, 2, 3, 4, 5, 6, 7 ormore generation to produce a tomato plant derived from the hybrid tomatovariety HM5235.

In some embodiments, the present invention teaches plants grown from thedeposited HM5235 seed wherein said plants comprise a single locusconversion. As used herein, the term “a” or “an” refers to one or moreof that entity; for example, “a single locus conversion” refers to oneor more single locus conversions or at least one single locusconversion. As such, the terms “a” (or “an”), “one or more” and “atleast one” are used interchangeably herein. In addition, reference to“an element” by the indefinite article “a” or “an” does not exclude thepossibility that more than one of the elements are present, unless thecontext clearly requires that there is one and only one of the elements.In some embodiments said single locus conversion confers said plantswith a trait selected from the group consisting of male sterility, malefertility, herbicide resistance, insect resistance, resistance forbacterial, fungal, mycoplasma or viral disease, enhanced plant qualitysuch as improved drought or salt tolerance, water stress tolerance,improved standability, enhanced plant vigor, improved shelf life,delayed senescence or controlled ripening, increased nutritional qualitysuch as increased sugar content or increased sweetness, increasedtexture, flavor and aroma, improved fruit length and/or size, protectionfor color, fruit shape, uniformity, length or diameter, refinement ordepth lodging resistance, yield and recovery when compared to a suitablecheck plant. In some embodiments, the check plant is a tomato hybridHM5235 not having said single locus conversion. In some embodiments, theat least one single locus conversion is an artificially mutated gene ora gene or nucleotide sequence modified through the use of New BreedingTechniques.

In some embodiments, the present invention provides a method ofproducing a commodity plant product comprising collecting the commodityplant product from the plant of the present invention. The commodityplant product produced by said method is also part of the presentinvention.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

In the description and tables that follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. An allele is any of one or more alternative forms of a genewhich relate to one trait or characteristic. In a diploid cell ororganism, the two alleles of a given gene occupy corresponding loci on apair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Commodity plant product. A “commodity plant product” refers to anycomposition or product that is comprised of material derived from aplant, seed, plant cell, or plant part of the present invention.Commodity plant products may be sold to consumers and can be viable ornonviable. Nonviable commodity products include but are not limited tononviable seeds and grains; processed seeds, seed parts, and plantparts; dehydrated plant tissue, frozen plant tissue, and processed planttissue; seeds and plant parts processed for animal feed for terrestrialand/or aquatic animal consumption, oil, meal, flour, flakes, bran,fiber, paper, tea, coffee, silage, crushed of whole grain, and any otherfood for human or animal consumption; and biomasses and fuel products;and raw material in industry.

Collection of seeds. In the context of the present invention acollection of seeds is a grouping of seeds mainly containing similarkind of seeds, for example hybrid seeds having the inbred line of theinvention as a parental line, but that may also contain, mixed togetherwith this first kind of seeds, a second, different kind of seeds, of oneof the inbred parent lines, for example the inbred line of the presentinvention. A commercial bag of hybrid seeds having the inbred line ofthe invention as a parental line and containing also the inbred lineseeds of the invention would be, for example such a collection of seeds.

Determinate tomatoes. Determinate tomatoes are tomato varieties thatcome to fruit all at once, then stop bearing. They are best suited forcommercial growing and mechanical harvesting since they can be harvestedall at once.

Decreased vigor. A plant having a decreased vigor in the presentinvention is a plant that, compared to other plants has a less vigorousappearance for vegetative and/or reproductive characteristics includingshorter plant height, small fruit size, fewer fruit or othercharacteristics

Enhanced nutritional quality. The nutritional quality of the tomato ofthe present invention can be enhanced by the introduction of severaltraits comprising a higher endosperm sugar content, flesh texture, brix,aroma content and increased sweetness, increased lycopene content of thepeel, etc.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics of the recurrent parent, except for the characteristicsderived from the converted gene.

Flesh color: In the context of the present invention, the flesh color isthe color of the tomato flesh that can range from orange-red to dark redwhen at ripe stage (harvest maturity).

Field holding ability: Field holding ability is the ability for fruitquality to maintain even after fruit is ripe (has turned red).

Grafting. Grafting is the operation by which a rootstock is grafted witha scion. The primary motive for grafting is to avoid damages bysoil-born pest and pathogens when genetic or chemical approaches fordisease management are not available. Grafting a susceptible scion ontoa resistant rootstock can provide a resistant cultivar without the needto breed the resistance into the cultivar. In addition, grafting mayenhance tolerance to abiotic stress, increase yield and result in moreefficient water and nutrient uses.

Immunity to disease(s) and or insect(s). A tomato plant which is notsubject to attack or infection by specific disease(s) and or insect(s)is considered immune.

Industrial usage. The industrial usage of the tomato of the presentinvention comprises the use of the tomato fruit for consumption, whetheras fresh products or in canning, freezing or any other industries.

Intermediate resistance to disease(s) and or insect(s). A tomato plantthat restricts the growth and development of specific disease(s) and orinsect(s), but may exhibit a greater range of symptoms or damagecompared to high resistant plants. Intermediate resistant plants willusually show less severe symptoms or damage than susceptible plantvarieties when grown under similar environmental conditions and/orspecific disease(s) and or insect(s) pressure, but may have heavy damageunder heavy pressure. Intermediate resistant tomato plants are notimmune to the disease(s) and or insect(s).

Maturity. In the region of best adaptability, maturity is the number ofdays from transplanting to optimal time for fruit harvest.

New Breeding Techniques: New breeding techniques are said of various newtechnologies developed and/or used to create new characteristics inplants through genetic variation, the aim being targeted mutagenesis,targeted introduction of new genes or gene silencing (RdDM). Example ofsuch new breeding techniques are targeted sequence changes facilitatedthru the use of Zinc finger nuclease (ZFN) technology (ZFN-1, ZFN-2 andZFN-3, see U.S. Pat. No. 9,145,565, incorporated by reference in itsentirety), Oligonucleotide directed mutagenesis (ODM), Cisgenesis andintragenesis, RNA-dependent DNA methylation (RdDM, which does notnecessarily change nucleotide sequence but can change the biologicalactivity of the sequence), Grafting (on GM rootstock), Reverse breeding,Agro-infiltration (agro-infiltration “sensu stricto”, agro-inoculation,floral dip), Transcription Activator-Like Effector Nucleases (TALENs,see U.S. Pat. Nos. 8,586,363 and 9,181,535, incorporated by reference intheir entireties), the CRISPR/Cas system (see U.S. Pat. Nos. 8,697,359;8,771,945; 8,795,965; 8,865,406; 8,871,445; 8,889,356; 8,895,308;8,906,616; 8,932,814; 8,945,839; 8,993,233; and 8,999,641, which are allhereby incorporated by reference), engineered meganuclease re-engineeredhoming endonucleases, DNA guided genome editing (Gao et al., NatureBiotechnology (2016), doi: 10.1038/nbt.3547, incorporated by referencein its entirety), and Synthetic genomics). A major part of today'stargeted genome editing, another designation for New BreedingTechniques, is the applications to induce a DNA double strand break(DSB) at a selected location in the genome where the modification isintended. Directed repair of the DSB allows for targeted genome editing.Such applications can be utilized to generate mutations (e.g., targetedmutations or precise native gene editing) as well as precise insertionof genes (e.g., cisgenes, intragenes, or transgenes). The applicationsleading to mutations are often identified as site-directed nuclease(SDN) technology, such as SDN1, SDN2 and SDN3. For SDN1, the outcome isa targeted, non-specific genetic deletion mutation: the position of theDNA DSB is precisely selected, but the DNA repair by the host cell israndom and results in small nucleotide deletions, additions orsubstitutions. For SDN2, a SDN is used to generate a targeted DSB and aDNA repair template (a short DNA sequence identical to the targeted DSBDNA sequence except for one or a few nucleotide changes) is used torepair the DSB: this results in a targeted and predetermined pointmutation in the desired gene of interest. As to the SDN3, the SDN isused along with a DNA repair template that contains new DNA sequence(e.g. gene). The outcome of the technology would be the integration ofthat DNA sequence into the plant genome. The most likely applicationillustrating the use of SDN3 would be the insertion of cisgenic,intragenic, or transgenic expression cassettes at a selected genomelocation. A complete description of each of these techniques can befound in the report made by the Joint Research Center (JRC) Institutefor Prospective Technological Studies of the European Commission in 2011and titled “New plant breeding techniques—State-of-the-art and prospectsfor commercial development”, which is incorporated by reference in itsentirety.

Plant adaptability. A plant having good plant adaptability means a plantthat will perform well in different growing conditions and seasons.

Plant Cell. As used herein, the term “plant cell” includes plant cellswhether isolated, in tissue culture or incorporated in a plant or plantpart.

Plant Part. As used herein, the term plant includes plant cells, plantprotoplasts, plant cell tissue cultures from which tomato plants can beregenerated, plant calli, plant clumps and plant cells that are intactin plants or parts of plants, such as embryos, pollen, ovules, flowers,seeds, fruit, rootstock, scions, stems, roots, anthers, pistils, roottips, leaves, meristematic cells, axillary buds, hypocotyls cotyledons,ovaries, seed coat endosperm and the like. In some embodiments, theplant part at least comprises at least one cell of said plant. In someembodiments, the plant part is further defined as a pollen, a meristem,a cell or an ovule.

Predicted paste bostwick. The predicted paste bostwick is the calculatednumber with the brix and Bostwick reading using the following formula:Predicted paste bostwick=−11.53+(1.64*juice brix)+(0.5*juice bostwick).

Quantitative Trait Loci (QTL) Quantitative trait loci refer to geneticloci that control to some degree numerically representable traits thatare usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Resistance to disease(s) and or insect(s). A tomato plant that restrictsthe growth and development of specific disease(s) and or insect(s) undernormal disease(s) and or insect(s) attack pressure when compared tosusceptible plants. These tomato plants can exhibit some symptoms ordamage under heavy disease(s) and or insect(s) pressure. Resistanttomato plants are not immune to the disease(s) and or insect(s).

Rootstock. A rootstock is the lower part of a plant capable of receivinga scion in a grafting process.

Scion. A scion is the higher part of a plant capable of being graftedonto a rootstock in a grafting process.

Semi-erect habit. A semi-erect plant has a combination of lateral andupright branching and has an intermediate type habit between a prostateplant habit, having laterally growing branching with fruits most of thetime on the ground and an erect plant habit with branching goingstraight up with fruit being off the ground.

Single gene converted (conversion). Single gene converted (conversion)plants refer to plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of a plant are recovered in additionto the single gene transferred into the plant via the backcrossingtechnique or via genetic engineering. A single gene converted plant canalso be referred to a plant obtained though mutagenesis or through theuse of some new breeding techniques, whereas the single gene convertedplant has essentially all of the desired morphological and physiologicalcharacteristics of the original variety in addition to the single geneor nucleotide sequence muted or engineered through the New BreedingTechniques.

Soluble Solids. Soluble solids refers to the percent of solid materialthat dissolve into tomato puree or juice, the vast majority of which issugars. Soluble solids are directly related to finished processedproduct yield of paste and sauce. Soluble solids are estimated with arefractometer, and measured as degrees brix.

Susceptible to disease(s) and or insect(s). A tomato plant that issusceptible to disease(s) and or insect(s) is defined as a tomato plantthat has the inability to restrict the growth and development ofspecific disease(s) and or insect(s). Plants that are susceptible willshow damage when infected and are more likely to have heavy damage undermoderate levels of specific disease(s) and or insect(s).

Tolerance to abiotic stresses. A tomato plant that is tolerant toabiotic stresses has the ability to endure abiotic stress withoutserious consequences for growth, appearance and yield.

Uniformity. Uniformity, as used herein, describes the similarity betweenplants or plant characteristics which can be a described by qualitativeor quantitative measurements.

Variety. A plant variety as used by one skilled in the art of plantbreeding means a plant grouping within a single botanical taxon of thelowest known rank which can be defined by the expression of thecharacteristics resulting from a given genotype or combination ofphenotypes, distinguished from any other plant grouping by theexpression of at least one of the said characteristics and considered asa unit with regard to its suitability for being propagated unchanged(International convention for the protection of new varieties ofplants).

Yield (Tomato yield Tons/Acre). The yield in tons/acre is the actualyield of the tomato fruit at harvest

Tomato Plants

Practically speaking, all cultivated and commercial forms of tomatobelong to a species most frequently referred to as Lycopersiconesculentum Miller. Lycopersicon is a relatively small genus within theextremely large and diverse family Solanaceae which is considered toconsist of around 90 genera, including pepper, tobacco and eggplant. Thegenus Lycopersicon has been divide into two subgenera, the esculentumcomplex which contains those species that can easily be crossed with thecommercial tomato and the peruvianum complex which contains thosespecies which are crossed with considerable difficulty (Stevens, M., andRick, C. M. 1986. Genetics and Breeding. In: The Tomato Crop. Ascientific basis for improvement, pp. 35-109. Atherton, J., Rudich, G.(eds.). Chapman and Hall, New York). Due to its value as a crop, L.esculentum Miller has become widely disseminated all over the world.Even if the precise origin of the cultivated tomato is still somewhatunclear, it seems to come from the Americas, being native to Ecuador,Peru and the Galapagos Island and initially cultivated by Aztecs andIncas as early as 700 AD. Mexico appears to have been the site ofdomestication and the source of the earliest introduction.

It is supposed that the cherry tomato, L. esculentum var. cerasiforme,is the direct ancestor of modern cultivated forms.

Tomato is grown for its fruit, widely used as a fresh market orprocessed product. As a crop, tomato is grown commercially whereverenvironmental conditions permit the production of an economically viableyield. In California, the first largest processing tomato market andsecond largest fresh market in the United States, processing tomato areharvested by machine. The majority of fresh market tomatoes areharvested by hand at vine ripe and mature green stage of ripeness. Freshmarket tomatoes are available in the United States year round.Processing tomato season in California is from late June to October.Processing tomato are used in many forms, as canned tomatoes, tomatojuice, tomato sauce, puree, paste or even catsup. Over the 500,000 acresof tomatoes that are grown annually in the US, approximately 40% aregrown for fresh market consumption, the balance are grown forprocessing.

Tomato is a normally simple diploid species with twelve pairs ofdifferentiated chromosomes. However, polyploidy tomato is also part ofthe present invention. The cultivated tomato is self-fertile and almostexclusively self-pollinating. The tomato flowers are hermaphrodites.Commercial cultivars were initially open pollinated. Most have now beenreplaced by better yielding hybrids. Due to its wide dissemination andhigh value, tomato has been intensively bred. This explains why such awide array of tomato is now available. The shape may range from small tolarge, and there are cherry, plum, pear, blocky, round, and beefsteaktypes. Tomatoes may be grouped by the amount of time it takes for theplants to mature fruit for harvest and, in general, the cultivars areconsidered to be early, midseason or late-maturing. Tomatoes can also begrouped by the plant's growth habit; determinate or indeterminate.Determinate plants tend to grow their foliage first, then set flowersthat mature into fruit if pollination is successful. All of the fruitstend to ripen on a plant at about the same time. Indeterminate tomatoesstart out by growing some foliage, then continue to produce foliage andflowers throughout the growing season. These plants will tend to havetomato fruit in different stages of maturity at any given time. Morerecent developments in tomato breeding have led to a wider array offruit color. In addition to the standard red ripe color, tomatoes can becreamy white, lime green, pink, yellow, golden, orange or purple.

Hybrid vigor has been documented in tomatoes and hybrids are gainingmore and more popularity amongst farmers with uniformity of plantcharacteristics.

Hybrid commercial tomato seed can be produced by hand pollination.Pollen of the male parent is harvested and manually applied to thestigmatic surface of the female inbred. Prior to and after handpollination, flowers are covered so that insects do not bring foreignpollen and create a mix or impurity. Flowers are tagged to identifypollinated fruit from which seed will be harvested.

There are numerous steps in the development of any novel, desirableplant germplasm. Plant breeding begins with the analysis and definitionof problems and weaknesses of the current germplasm, the establishmentof program goals, and the definition of specific breeding objectives.The next step is selection of germplasm that possesses the traits tomeet the program goals. The goal is to combine in a single variety orhybrid an improved combination of desirable traits from the parentalgermplasm.

In tomato, these important traits may include increased fruit number,fruit size and fruit weight, higher seed yield, improved color,resistance to diseases and insects, tolerance to drought and heat,better uniformity, higher nutritional value and better agronomicquality, growth rate, high seed germination, seedling vigor, early fruitmaturity, ease of fruit setting, adaptability for soil and climateconditions, firmness, content in soluble solids, acidity and viscosity.With mechanical harvesting of processing tomato, fruit settingconcentration, harvestability and field holding are also very important.

In some embodiments, particularly desirable traits that may beincorporated by this invention are improved resistance to differentviral, fungal, and bacterial pathogens. Important diseases include butare not limited to Tomato yellow leaf curl virus, Tomato spot wiltvirus, etc. Improved resistance to insect pests is another desirabletrait that may be incorporated into new tomato plants developed by thisinvention. Insect pests affecting the various species of tomato include,but not limited to arthropod pests such as Tuta absoluta, Frankliniellaoccidentalis, Bemisia tabaci, etc.

Other desirable traits include traits related to improved tomato fruits.A non-limiting list of fruit phenotypes used during breeding selectioninclude:

-   -   Average of juice bostwick. The juice Bostwick a measurement of        the viscosity. The viscosity or consistency of tomato products        is affected by the degree of concentration of the tomato, the        amount of and extent of degradation of pectin, the size, shape        and quality of the pulp, and probably to a lesser extent, by the        proteins, sugars and other soluble constituents. The viscosity        is measured in Bostwick centimeters by using instruments such as        a Bostwick Consistometer.    -   pH. The pH is a measure of acidity of the fruit puree. A pH        under 4.5 is desirable to prevent bacterial spoilage of finished        products. pH rises as fruit matures.    -   Fruit color. Fruit color is measured as Hunters a/b ratio, where        a represents red/green, positive values are red, negative values        are green and 0 is neutral; b represents yellow/blue, where        positive values are yellow, negative values are blue and 0 is        neutral; a/b represents the intense of redness: large value        represents deep red color, small value represents light or        yellowish red color.    -   Fruit Weight. The weight of a single fruit or the average of        many fruit measured at harvest maturity and recorded in a        convenient unit of measure.    -   Ostwald. The Ostwald is a measurement of serum viscosity whereas        the measurement are taken using an Ostwald viscometer. The serum        is the non-solid portion of a tomato extract after        centrifugation of the tomato puree. The serum viscosity is        affected by the quantity and quality of soluble pectin. Higher        number reflect higher viscosity of the tomato serum.    -   Fruit firmness. The fruit firmness is the resistance to        penetration and is measured using a Digital Durometer Model        DD-4-00 (Rex Gauge Company, Buffalo Grove, Ill., USA). Durometer        readings are taken at 4 locations (each about 90 degrees apart)        on the approximate mid-point of a tomato, with the tomato laying        on its side. From a fruit sample collected at a given location,        the resistance to penetration is measured with the durometer        from 9 individual fruit at 4 locations per fruit (a total of 36        independent measurements). The P5 value is calculated from the        following equation: D-39/10, where D is the value from the        Durometer.        Tomato Breeding

The goal of tomato breeding is to develop new, unique and superiortomato inbred lines and hybrids. The breeder initially selects andcrosses two or more parental lines, followed by repeated selfing andselection, producing many new genetic combinations. Another method usedto develop new, unique and superior tomato inbred lines and hybridsoccurs when the breeder selects and crosses two or more parental linesfollowed by haploid induction and chromosome doubling that result in thedevelopment of dihaploid inbred lines. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations and the same is true for the utilization of thedihaploid breeding method.

During the development of new tomato inbreds and hybrids, the tomatobreeder uses pepper plants, but also non-commercial tomato plants, suchas plants that may contain characteristics that the breeder has interestin having in its tomato inbreds and hybrids. Such non-commercial tomatoplants could be wild relatives of tomato species.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred linesdeveloped are unpredictable. This unpredictability is because thebreeder's selection occurs in unique environments, with no control atthe DNA level (using conventional breeding procedures or dihaploidbreeding procedures), and with millions of different possible geneticcombinations being generated. A breeder of ordinary skill in the artcannot predict the final resulting lines he develops, except possibly ina very broad and general fashion. This unpredictability results in theexpenditure of large research monies to develop superior new tomatoinbred lines and hybrids.

The development of commercial tomato hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the F1 hybrid crosses.

Pedigree breeding and recurrent selection breeding methods are used todevelop inbred lines from breeding populations. Breeding programscombine desirable traits from two or more inbred lines or variousbroad-based sources into breeding pools from which inbred lines aredeveloped by selfing and selection of desired phenotypes or through thedihaploid breeding method followed by the selection of desiredphenotypes. The new inbreds are crossed with other inbred lines and thehybrids from these crosses are evaluated to determine which havecommercial potential.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, recurrent selection, andbackcross breeding.

i Pedigree Selection

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents possessing favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁s or by intercrossing two F₁s (sib mating). The dihaploid breedingmethod could also be used. Selection of the best individuals is usuallybegun in the F₂ population; then, beginning in the F₃, the bestindividuals in the best families are selected. Replicated testing offamilies, or hybrid combinations involving individuals of thesefamilies, often follows in the F₄ generation to improve theeffectiveness of selection for traits with low heritability. At anadvanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potential use asparents of new hybrid cultivars. Similarly, the development of newinbred lines through the dihaploid system requires the selection of thebest inbreds followed by two to five years of testing in hybridcombinations in replicated plots.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F2 to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F2 individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F2 plantsoriginally sampled in the population will be represented by a progenywhen generation advance is completed.

In a multiple-seed procedure, breeders commonly harvest one or morefruit containing seed from each plant in a population and blend themtogether to form a bulk seed lot. Part of the bulked seed is used toplant the next generation and part is put in reserve. The procedure hasbeen referred to as modified single-seed descent or the bulk technique.

The multiple-seed procedure has been used to save labor at harvest. Itis considerably faster than removing one seed from each fruit by handfor the single seed procedure. The multiple-seed procedure also makes itpossible to plant the same number of seeds of a population eachgeneration of inbreeding. Enough seeds are harvested to make up forthose plants that did not germinate or produce seed.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., R. W. Allard, 1960, Principles of Plant Breeding, JohnWiley and Son, pp. 115-161; N. W. Simmonds, 1979, Principles of CropImprovement, Longman Group Limited; W. R. Fehr, 1987, Principles of CropDevelopment, Macmillan Publishing Co.; N. F. Jensen, 1988, PlantBreeding Methodology, John Wiley & Sons).

ii Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype recurrent parent andthe trait of interest from the donor parent are selected and repeatedlycrossed (backcrossed) to the recurrent parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent.

When the term hybrid tomato plant is used in the context of the presentinvention, this also includes any hybrid tomato plant where one or moredesired trait has been introduced through backcrossing methods, whethersuch trait is a naturally occurring one, a mutant, a transgenic one or agene or a nucleotide sequence modified by the use of New BreedingTechniques. Backcrossing methods can be used with the present inventionto improve or introduce one or more characteristic into the inbredparental line, thus potentially introducing these traits in to thehybrid tomato plant of the present invention. The term “backcrossing” asused herein refers to the repeated crossing of a hybrid progeny back tothe recurrent parent, i.e., backcrossing one, two, three, four, five,six, seven, eight, nine, or more times to the recurrent parent. Theparental tomato plant which contributes the gene or the genes for thedesired characteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental tomato plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol.

In a typical backcross protocol, the original inbred of interest(recurrent parent) is crossed to a second inbred (nonrecurrent parent)that carries the gene or genes of interest to be transferred. Theresulting progeny from this cross are then crossed again to therecurrent parent and the process is repeated until a tomato plant isobtained wherein all the desired morphological and physiologicalcharacteristics of the recurrent parent are recovered in the convertedplant, generally determined at a 5% significance level when grown in thesame environmental conditions, in addition to the gene or genestransferred from the nonrecurrent parent. It has to be noted that some,one, two, three or more, self-pollination and growing of populationmight be included between two successive backcrosses. Indeed, anappropriate selection in the population produced by theself-pollination, i.e. selection for the desired trait and physiologicaland morphological characteristics of the recurrent parent might beequivalent to one, two or even three additional backcrosses in acontinuous series without rigorous selection, saving then time, moneyand effort to the breeder. A non-limiting example of such a protocolwould be the following: a) the first generation F1 produced by the crossof the recurrent parent A by the donor parent B is backcrossed to parentA, b) selection is practiced for the plants having the desired trait ofparent B, c) selected plant are self-pollinated to produce a populationof plants where selection is practiced for the plants having the desiredtrait of parent B and physiological and morphological characteristics ofparent A, d) the selected plants are backcrossed one, two, three, four,five, six, seven, eight, nine, or more times to parent A to produceselected backcross progeny plants comprising the desired trait of parentB and the physiological and morphological characteristics of parent A.Step (c) may or may not be repeated and included between the backcrossesof step (d).

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute one or more trait(s) or characteristic(s) in theoriginal inbred parental line in order to find it then in the hybridmade thereof. To accomplish this, a gene or genes of the recurrentinbred is modified or substituted with the desired gene or genes fromthe nonrecurrent parent, while retaining essentially all of the rest ofthe desired genetic, and therefore the desired physiological andmorphological, constitution of the original inbred. The choice of theparticular nonrecurrent parent will depend on the purpose of thebackcross; one of the major purposes is to add some commerciallydesirable, agronomically important trait(s) to the plant. The exactbackcrossing protocol will depend on the characteristic(s) or trait(s)being altered to determine an appropriate testing protocol. Althoughbackcrossing methods are simplified when the characteristic beingtransferred is a single gene and dominant allele, multiple genes andrecessive allele(s) may also be transferred and therefore, backcrossbreeding is by no means restricted to character(s) governed by one or afew genes. In fact the number of genes might be less important that theidentification of the character(s) in the segregating population. Inthis instance it may then be necessary to introduce a test of theprogeny to determine if the desired characteristic(s) has beensuccessfully transferred. Such tests encompass visual inspection, simplecrossing, but also follow up of the characteristic(s) throughgenetically associated markers and molecular assisted breeding tools.For example, selection of progeny containing the transferred trait isdone by direct selection, visual inspection for a trait associated witha dominant allele, while the selection of progeny for a trait that istransferred via a recessive allele, such as the orange fruit colorcharacteristic in tomato, requires selfing the progeny or usingmolecular markers to determine which plant carry the recessiveallele(s).

Many single gene traits have been identified that are not regularlyselected for in the development of a new parental inbred of a hybridtomato plant according to the invention but that can be improved bybackcrossing techniques. Single gene traits may or may not betransgenic. Examples of these traits include but are not limited to,male sterility (such as the ms1, ms2, ms3, ms4 or ms5 genes), herbicideresistance (such as bar or PAT genes), resistance for bacterial, fungal(genes Cf for resistance to Cladosporium fulvum), or viral disease (geneTy for resistance to Tomato Yellow Leaf Curl Virus (TYLCV), genes Tm-1,Tm-2 and Tm2² for the resistance to the tomato mosaic tobamovirus(ToMV)), insect resistance (gene Mi for resistance to nematodes),increased brix by introduction of specific alleles such as the hir4allele from lycopersicon hirsutum, high lycopene by using the dg mutantas described in U.S. Ser. No. 10/587,789, improved shelf life by usingmutants such as the rin (ripening inhibitor), nor (non-ripening) or cnr(colorless non ripening) alleles, increased firmness or slower softeningof the fruits due, for example in a mutation in an expansin gene,absence of gel (i.e. fruits having a cavity area which is solid andlacks a gel or liquid content male) by the use of the PSAF allele,fertility, enhanced nutritional quality, enhanced sugar content, yieldstability and yield enhancement. These genes are generally inheritedthrough the nucleus. Some known exceptions to this are the genes formale sterility, some of which are inherited cytoplasmically, but stillact as single gene traits. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

In 1981, the backcross method of breeding counted for 17% of the totalbreeding effort for inbred line development in the United States,accordingly to, Hallauer, A. R. et al. (1988) “Corn Breeding” Corn andCorn Improvement, No. 18, pp. 463-481.

The backcross breeding method provides a precise way of improvingvarieties that excel in a large number of attributes but are deficientin a few characteristics. (Page 150 of the Pr. R. W. Allard's 1960 book,published by John Wiley & Sons, Inc., Principles of Plant Breeding). Themethod makes use of a series of backcrosses to the variety to beimproved during which the character or the characters in whichimprovement is sought is maintained by selection. At the end of thebackcrossing the gene or genes being transferred unlike all other genes,will be heterozygous. Selfing after the last backcross produceshomozygosity for this gene pair(s) and, coupled with selection, willresult in a parental line of a hybrid variety with exactly oressentially the same adaptation, yielding ability and qualitycharacteristics of the recurrent parent but superior to that parent inthe particular characteristic(s) for which the improvement program wasundertaken. Therefore, this method provides the plant breeder with ahigh degree of genetic control of his work.

The method is scientifically exact because the morphological andagricultural features of the improved variety could be described inadvance and because a similar variety could, if it were desired, be breda second time by retracing the same steps (Briggs, “Breeding wheatsresistant to bunt by the backcross method”, 1930 Jour. Amer. Soc.Agron., 22: 289-244).

Backcrossing is a powerful mechanism for achieving homozygosity and anypopulation obtained by backcrossing must rapidly converge on thegenotype of the recurrent parent. When backcrossing is made the basis ofa plant breeding program, the genotype of the recurrent parent will betheoretically modified only with regards to genes being transferred,which are maintained in the population by selection.

Successful backcrosses are, for example, the transfer of stem rustresistance from ‘Hope’ wheat to ‘Bart wheat’ and even pursuing thebackcrosses with the transfer of bunt resistance to create ‘Bart 38’,having both resistances. Also highlighted by Allard is the successfultransfer of mildew, leaf spot and wilt resistances in California Commonalfalfa to create ‘Caliverde’. This new ‘Caliverde’ variety producedthrough the backcross process is indistinguishable from CaliforniaCommon except for its resistance to the three named diseases.

One of the advantages of the backcross method is that the breedingprogram can be carried out in almost every environment that will allowthe development of the character being transferred or when usingmolecular markers that can identify the trait of interest.

The backcross technique is not only desirable when breeding for diseaseresistance but also for the adjustment of morphological characters,color characteristics and simply inherited quantitative characters suchas earliness, plant height and seed size and shape. In this regard, amedium grain type variety, ‘Calady’, has been produced by Jones andDavis. As dealing with quantitative characteristics, they selected thedonor parent with the view of sacrificing some of the intensity of thecharacter for which it was chosen, i.e. grain size. ‘Lady Wright’, along grain variety was used as the donor parent and ‘Coloro’, a shortgrain one as the recurrent parent. After four backcrosses, the mediumgrain type variety ‘Calady’ was produced.

iii Open-Pollinated Populations

The improvement of open-pollinated populations of such crops as rye,many maizes and sugar beets, herbage grasses, legumes such as alfalfaand clover, and tropical tree crops such as cacao, coconuts, oil palmand some rubber, depends essentially upon changing gene-frequenciestowards fixation of favorable alleles while maintaining a high (but farfrom maximal) degree of heterozygosity.

Uniformity in such populations is impossible and trueness-to-type in anopen-pollinated variety is a statistical feature of the population as awhole, not a characteristic of individual plants. Thus, theheterogeneity of open-pollinated populations contrasts with thehomogeneity (or virtually so) of inbred lines, clones and hybrids.

Population improvement methods fall naturally into two groups, thosebased on purely phenotypic selection, normally called mass selection,and those based on selection with progeny testing. Interpopulationimprovement utilizes the concept of open breeding populations; allowinggenes to flow from one population to another. Plants in one population(cultivar, strain, ecotype, or any germplasm source) are crossed eithernaturally (e.g., by wind) or by hand or by bees (commonly Apis melliferaL. or Megachile rotundata F.) with plants from other populations.Selection is applied to improve one (or sometimes both) population(s) byisolating plants with desirable traits from both sources.

There are basically two primary methods of open-pollinated populationimprovement.

First, there is the situation in which a population is changed en masseby a chosen selection procedure. The outcome is an improved populationthat is indefinitely propagated by random-mating within itself inisolation.

Second, the synthetic variety attains the same end result as populationimprovement, but is not itself propagated as such; it has to bereconstructed from parental lines or clones. These plant breedingprocedures for improving open-pollinated populations are well known tothose skilled in the art and comprehensive reviews of breedingprocedures routinely used for improving cross-pollinated plants areprovided in numerous texts and articles, including: Allard, Principlesof Plant Breeding, John Wiley & Sons, Inc. (1960); Simmonds, Principlesof Crop Improvement, Longman Group Limited (1979); Hallauer and Miranda,Quantitative Genetics in Maize Breeding, Iowa State University Press(1981); and, Jensen, Plant Breeding Methodology, John Wiley & Sons, Inc.(1988).

A) Mass Selection

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued. In massselection, desirable individual plants are chosen, harvested, and theseed composited without progeny testing to produce the followinggeneration. Since selection is based on the maternal parent only, andthere is no control over pollination, mass selection amounts to a formof random mating with selection. As stated above, the purpose of massselection is to increase the proportion of superior genotypes in thepopulation.

B) Synthetics

A synthetic variety is produced by intercrossing a number of genotypesselected for good combining ability in all possible hybrid combinations,with subsequent maintenance of the variety by open pollination. Whetherparents are (more or less inbred) seed-propagated lines, as in somesugar beet and beans (Vicia) or clones, as in herbage grasses, cloversand alfalfa, makes no difference in principle. Parents are selected ongeneral combining ability, sometimes by test crosses or toperosses, moregenerally by polycrosses. Parental seed lines may be deliberately inbred(e.g. by selfing or sib crossing). However, even if the parents are notdeliberately inbred, selection within lines during line maintenance willensure that some inbreeding occurs. Clonal parents will, of course,remain unchanged and highly heterozygous.

Whether a synthetic can go straight from the parental seed productionplot to the farmer or must first undergo one or more cycles ofmultiplication depends on seed production and the scale of demand forseed. In practice, grasses and clovers are generally multiplied once ortwice and are thus considerably removed from the original synthetic.

While mass selection is sometimes used, progeny testing is generallypreferred for polycrosses, because of their operational simplicity andobvious relevance to the objective, namely exploitation of generalcombining ability in a synthetic.

The number of parental lines or clones that enters a synthetic varieswidely. In practice, numbers of parental lines range from 10 to severalhundred, with 100-200 being the average. Broad based synthetics formedfrom 100 or more clones would be expected to be more stable during seedmultiplication than narrow based synthetics.

iv. Hybrids

A hybrid is an individual plant resulting from a cross between parentsof differing genotypes. Commercial hybrids are now used extensively inmany crops, including corn (maize), sorghum, sugarbeet, sunflowerbroccoli and tomato. Hybrids can be formed in a number of differentways, including by crossing two parents directly (single cross hybrids),by crossing a single cross hybrid with another parent (three-way ortriple cross hybrids), or by crossing two different hybrids (four-way ordouble cross hybrids).

Strictly speaking, most individuals in an out breeding (i.e.,open-pollinated) population are hybrids, but the term is usuallyreserved for cases in which the parents are individuals whose genomesare sufficiently distinct for them to be recognized as different speciesor subspecies. Hybrids may be fertile or sterile depending onqualitative and/or quantitative differences in the genomes of the twoparents. Heterosis, or hybrid vigor, is usually associated withincreased heterozygosity that results in increased vigor of growth,survival, and fertility of hybrids as compared with the parental linesthat were used to form the hybrid. Maximum heterosis is usually achievedby crossing two genetically different, highly inbred lines.

Hybrid commercial tomato seed is produced by controlled handpollination. The anthers of the female parent are removed and pollen ofthe male parent is harvested and manually applied to the stigmaticsurface of the female inbred. Prior to, and after hand pollination,flowers are covered so that insects do not bring foreign pollen andcreate a mix or impurity. Flowers are tagged to identify pollinatedfruit from which seed will be harvested.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F1progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F1 hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor and uniformity exhibited byF1 hybrids is lost in the next generation (F2). Consequently, seed fromF2 hybrid varieties is not used for planting stock.

The production of hybrids is a well-developed industry, involving theisolated production of both the parental lines and the hybrids whichresult from crossing those lines. For a detailed discussion of thehybrid production process, see, e.g., Wright, Commercial Hybrid SeedProduction 8:161-176, In Hybridization of Crop Plants.

v. Bulk Segregation Analysis (BSA)

BSA, a.k.a. bulked segregation analysis, or bulk segregant analysis, isa method described by Michelmore et al. (Michelmore et al., 1991,Identification of markers linked to disease-resistance genes by bulkedsegregant analysis: a rapid method to detect markers in specific genomicregions by using segregating populations. Proceedings of the NationalAcademy of Sciences, USA, 99:9828-9832) and Quarrie et al. (Quarrie etal., 1999, Journal of Experimental Botany, 50(337): 1299-1306).

For BSA of a trait of interest, parental lines with certain differentphenotypes are chosen and crossed to generate F2, doubled haploid orrecombinant inbred populations with QTL analysis. The population is thenphenotyped to identify individual plants or lines having high or lowexpression of the trait. Two DNA bulks are prepared, one from theindividuals having one phenotype (e.g., resistant to virus), and theother from the individuals having reversed phenotype (e.g., susceptibleto virus), and analyzed for allele frequency with molecular markers.Only a few individuals are required in each bulk (e.g., 10 plants each)if the markers are dominant (e.g., RAPDs). More individuals are neededwhen markers are co-dominant (e.g., RFLPs, SNPs or SSRs). Markers linkedto the phenotype can be identified and used for breeding or QTL mapping.

vi. Hand-Pollination Method

Hand pollination describes the crossing of plants via the deliberatefertilization of female ovules with pollen from a desired male parentplant. In some embodiments the donor or recipient female parent and thedonor or recipient male parent line are planted in the same field. Theinbred male parent can be planted earlier than the female parent toensure adequate pollen supply at the pollination time. In someembodiments, the male parent and female parent can be planted at a ratioof 1 male parent to 4-10 female parents. The male parent may be plantedat the top of the field for efficient male flower collection duringpollination. Pollination is started when the female parent flower isready to be fertilized. Female flower buds that are ready to open in thefollowing days are identified, covered with paper cups or small paperbags that prevent bee or any other insect from visiting the femaleflowers, and marked with any kind of material that can be easily seenthe next morning. In some embodiments, this process is best done in theafternoon. The male flowers of the male parent are collected in theearly morning before they are open and visited by pollinating insects.The covered female flowers of the female parent, which have opened, areun-covered and pollinated with the collected fresh male flowers of themale parent, starting as soon as the male flower sheds pollen. Thepollinated female flowers are again covered after pollination to preventbees and any other insects visit. The pollinated female flowers are alsomarked. The marked fruits are harvested. In some embodiments, the malepollen used for fertilization has been previously collected and stored.

vii. Bee-Pollination Method

Using the bee-pollination method, the parent plants are usually plantedwithin close proximity. In some embodiments more female plants areplanted to allow for a greater production of seed. Breeding of dioeciousspecies can also be done by growing equal amount of each parent plant.Insects are placed in the field or greenhouses for transfer of pollenfrom the male parent to the female flowers of the female parent.

viii. Targeting Induced Local Lesions in Genomes (TILLING)

Breeding schemes of the present application can include crosses withTILLING® plant lines. TILLING® is a method in molecular biology thatallows directed identification of mutations in a specific gene. TILLING®was introduced in 2000, using the model plant Arabidopsis thaliana.TILLING® has since been used as a reverse genetics method in otherorganisms such as zebrafish, corn, wheat, rice, soybean, tomato andlettuce.

The method combines a standard and efficient technique of mutagenesiswith a chemical mutagen (e.g., Ethyl methanesulfonate (EMS)) with asensitive DNA screening-technique that identifies single base mutations(also called point mutations) in a target gene. EcoTILLING is a methodthat uses TILLING® techniques to look for natural mutations inindividuals, usually for population genetics analysis (see Comai, etal., 2003 The Plant Journal 37, 778-786; Gilchrist et al. 2006 Mol.Ecol. 15, 1367-1378; Mejlhede et al. 2006 Plant Breeding 125, 461-467;Nieto et al. 2007 BMC Plant Biology 7, 34-42, each of which isincorporated by reference hereby for all purposes). DEcoTILLING is amodification of TILLING® and EcoTILLING which uses an inexpensive methodto identify fragments (Garvin et al., 2007, DEco-TILLING: An inexpensivemethod for SNP discovery that reduces ascertainment bias. MolecularEcology Notes 7, 735-746).

The TILLING® method relies on the formation of heteroduplexes that areformed when multiple alleles (which could be from a heterozygote or apool of multiple homozygotes and heterozygotes) are amplified in a PCR,heated, and then slowly cooled. As DNA bases are not pairing at themismatch of the two DNA strands (the induced mutation in TILLING® or thenatural mutation or SNP in EcoTILLING), they provoke a shape change inthe double strand DNA fragment which is then cleaved by single strandednucleases. The products are then separated by size on several differentplatforms.

Several TILLING® centers exists over the world that focus onagriculturally important species: UC Davis (USA), focusing on Rice;Purdue University (USA), focusing on Maize; University of BritishColumbia (CA), focusing on Brassica napus; John Innes Centre (UK),focusing on Brassica rapa; Fred Hutchinson Cancer Research, focusing onArabidopsis; Southern Illinois University (USA), focusing on Soybean;John Innes Centre (UK), focusing on Lotus and Medicago; and INRA(France), focusing on Pea and Tomato.

More detailed description on methods and compositions on TILLING® can befound in U.S. Pat. No. 5,994,075, US 2004/0053236 A1, WO 2005/055704,and WO 2005/048692, each of which is hereby incorporated by referencefor all purposes.

Thus in some embodiments, the breeding methods of the present disclosureinclude breeding with one or more TILLING plant lines with one or moreidentified mutations.

viii Mutation Breeding

Mutation breeding is another method of introducing new variation andsubsequent traits into tomato plants. Mutations that occur spontaneouslyor are artificially induced can be useful sources of variability for aplant breeder. The goal of artificial mutagenesis is to increase therate of mutation for a desired characteristic. Mutation rates can beincreased by many different means or mutating agents includingtemperature, long-term seed storage, tissue culture conditions,radiation (such as X-rays, Gamma rays, neutrons, Beta radiation, orultraviolet radiation), chemical mutagens (such as base analogs like5-bromo-uracil), antibiotics, alkylating agents (such as sulfurmustards, nitrogen mustards, epoxides, ethyleneamines, sulfates,sulfonates, sulfones, or lactones), azide, hydroxylamine, nitrous acidor acridines. Once a desired trait is observed through mutagenesis thetrait may then be incorporated into existing germplasm by traditionalbreeding techniques. Details of mutation breeding can be found in W. R.Fehr, 1993, Principles of Cultivar Development, Macmillan Publishing Co.

New breeding techniques such as the ones involving the uses of ZincFinger Nucleases or oligonucleotide directed mutagenesis shall also beused to generate genetic variability and introduce new traits intotomato varieties.

ix. Double Haploids and Chromosome Doubling

One way to obtain homozygous plants without the need to cross twoparental lines followed by a long selection of the segregating progeny,and/or multiple backcrossing is to produce haploids and then double thechromosomes to form doubled haploids. Haploid plants can occurspontaneously, or may be artificially induced via chemical treatments orby crossing plants with inducer lines (Seymour et al. 2012, PNAS vol.109, pg. 4227-4232; Zhang et al., 2008 Plant Cell Rep. December 27(12)1851-60). The production of haploid progeny can occur via a variety ofmechanisms which can affect the distribution of chromosomes duringgamete formation. The chromosome complements of haploids sometimesdouble spontaneously to produce homozygous doubled haploids (DHs).Mixoploids, which are plants which contain cells having differentploidies, can sometimes arise and may represent plants that areundergoing chromosome doubling so as to spontaneously produce doubledhaploid tissues, organs, shoots, floral parts or plants. Another commontechnique is to induce the formation of double haploid plants with achromosome doubling treatment such as colchicine (El-Hennawy et al.,2011 Vol 56, issue 2 pg. 63-72; Doubled Haploid Production in CropPlants 2003 edited by Maluszynski ISBN 1-4020-1544-5). The production ofdoubled haploid plants yields highly uniform inbred lines and isespecially desirable as an alternative to sexual inbreeding oflonger-generation crops. By producing doubled haploid progeny, thenumber of possible gene combinations for inherited traits is moremanageable. Thus, an efficient doubled haploid technology cansignificantly reduce the time and the cost of inbred and cultivardevelopment.

x. Protoplast Fusion

In another method for breeding plants, protoplast fusion can also beused for the transfer of trait-conferring genomic material from a donorplant to a recipient plant. Protoplast fusion is an induced orspontaneous union, such as a somatic hybridization, between two or moreprotoplasts (cells of which the cell walls are removed by enzymatictreatment) to produce a single bi- or multi-nucleate cell. The fusedcell that may even be obtained with plant species that cannot beinterbred in nature is tissue cultured into a hybrid plant exhibitingthe desirable combination of traits.

xi. Embryo Rescue

Alternatively, embryo rescue may be employed in the transfer ofresistance-conferring genomic material from a donor plant to a recipientplant. Embryo rescue can be used as a procedure to isolate embryos fromcrosses to rapidly move to the next generation of backcrossing orselfing or wherein plants fail to produce viable seed. In this process,the fertilized ovary or immature seed of a plant is tissue cultured tocreate new plants (see Pierik, 1999, In Vitro Culture of Higher Plants,Springer, ISBN 079235267x, 9780792352679, which is incorporated hereinby reference in its entirety).

Grafting

Grafting is a process that has been used for many years in crops such ascucurbitacea, but only more recently for tomato. Grafting may be used toprovide a certain level of resistance to telluric pathogens such asPhytophthora or to certain nematodes. Grating is therefore intended toprevent contact between the plant or variety to be cultivated and theinfested soil. The variety of interest used as the graft or scion,optionally an F1 hybrid, is grafted onto the resistant plant used as therootstock. The resistant rootstock remains healthy and provides, fromthe soils, the normal supply for the graft that it isolates from thediseases. In some recent developments, it has also been shown that somerootstocks are also able to improve the agronomic value for the graftedplant and in particular the equilibrium between the vegetative andgenerative development that are always difficult to balance in peppercultivation.

Breeding Evaluation

Each breeding program can include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested per se and inhybrid combination and compared to appropriate standards in environmentsrepresentative of the commercial target area(s). The best lines arecandidates for use as parents in new commercial cultivars; those stilldeficient in a few traits may be used as parents to produce newpopulations for further selection or in a backcross program to improvethe parent lines for a specific trait.

In one embodiment, the plants are selected on the basis of one or morephenotypic traits. Skilled persons will readily appreciate that suchtraits include any observable characteristic of the plant, including forexample growth rate, vigor, plant health, maturity, branching, plantheight, leaf coverage, weight, color, taste, smell, changes in theproduction of one or more compounds by the plant (including for example,metabolites, proteins, drugs, carbohydrates, oils, and any othercompounds).

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

It should be appreciated that in certain embodiments, plants may beselected based on the absence, suppression or inhibition of a certainfeature or trait (such as an undesirable feature or trait) as opposed tothe presence of a certain feature or trait (such as a desirable featureor trait).

Selecting plants based on genotypic information is also envisaged (forexample, including the pattern of plant gene expression, genotype, orpresence of genetic markers). Where the presence of one or more geneticmarker is assessed, the one or more marker may already be known and/orassociated with a particular characteristic of a plant; for example, amarker or markers may be associated with an increased growth rate ormetabolite profile. This information could be used in combination withassessment based on other characteristics in a method of the disclosureto select for a combination of different plant characteristics that maybe desirable. Such techniques may be used to identify novel quantitativetrait loci (QTLs). By way of example, plants may be selected based ongrowth rate, size (including but not limited to weight, height, leafsize, stem size, branching pattern, or the size of any part of theplant), general health, survival, tolerance to adverse physicalenvironments and/or any other characteristic, as described hereinbefore.

Further non-limiting examples include selecting plants based on: speedof seed germination; quantity of biomass produced; increased root,and/or leaf/shoot growth that leads to an increased yield (fruit) orbiomass production; effects on plant growth that results in an increasedseed yield for a crop; effects on plant growth which result in anincreased yield; effects on plant growth that lead to an increasedresistance or tolerance to disease including fungal, viral or bacterialdiseases, to mycoplasma, or to pests such as insects, mites or nematodesin which damage is measured by decreased foliar symptoms such as theincidence of bacterial or fungal lesions, or area of damaged foliage orreduction in the numbers of nematode cysts or galls on plant roots, orimprovements in plant yield in the presence of such plant pests anddiseases; effects on plant growth that lead to increased metaboliteyields; effects on plant growth that lead to improved aesthetic appealwhich may be particularly important in plants grown for their form,color or taste, for example the color intensity of tomato flesh, or thetaste of said fruit.

Molecular Breeding Evaluation Techniques

Selection of plants based on phenotypic or genotypic information may beperformed using techniques such as, but not limited to: high through-putscreening of chemical components of plant origin, sequencing techniquesincluding high through-put sequencing of genetic material, differentialdisplay techniques (including DDRT-PCR, and DD-PCR), nucleic acidmicroarray techniques, RNA-seq (Transcriptome Sequencing), qRTPCR(quantitative real time PCR).

In one embodiment, the evaluating step of a plant breeding programinvolves the identification of desirable traits in progeny plants.Progeny plants can be grown in, or exposed to conditions designed toemphasize a particular trait (e.g. drought conditions for droughttolerance, lower temperatures for freezing tolerant traits). Progenyplants with the highest scores for a particular trait may be used forsubsequent breeding steps.

In some embodiments, plants selected from the evaluation step canexhibit a 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 120% or more improvement in aparticular plant trait compared to a control plant.

In other embodiments, the evaluating step of plant breeding comprisesone or more molecular biological tests for genes or other markers. Forexample, the molecular biological test can involve probe hybridizationand/or amplification of nucleic acid (e.g., measuring nucleic aciddensity by Northern or Southern hybridization, PCR) and/or immunologicaldetection (e.g., measuring protein density, such as precipitation andagglutination tests, ELISA (e.g., Lateral Flow test or DAS-ELISA),Western blot, immune labeling, immunosorbent electron microscopy (ISEM),and/or dot blot).

The procedure to perform a nucleic acid hybridization, an amplificationof nucleic acid (e.g., PCR, RT-PCR) or an immunological detection (e.g.,precipitation and agglutination tests, ELISA (e.g., Lateral Flow test orDAS-ELISA), Western blot, RIA, immunogold or immunofluorescent labeling,immunosorbent electron microscopy (ISEM), and/or dot blot tests) areperformed as described elsewhere herein and well-known by one skilled inthe art.

In one embodiment, the evaluating step comprises PCR (semi-quantitativeor quantitative), wherein primers are used to amplify one or morenucleic acid sequences of a desirable gene, or a nucleic acid associatedwith said gene, or QTL or a desirable trait (e.g., a co-segregatingnucleic acid, or other marker).

In another embodiment, the evaluating step comprises immunologicaldetection (e.g., precipitation and agglutination tests, ELISA (e.g.,Lateral Flow test or DAS-ELISA), Western blot, RIA, immuno labeling(gold, fluorescent, or other detectable marker), immunosorbent electronmicroscopy (ISEM), and/or dot blot), wherein one or more gene ormarker-specific antibodies are used to detect one or more desirableproteins. In one embodiment, said specific antibody is selected from thegroup consisting of polyclonal antibodies, monoclonal antibodies,antibody fragments, and combination thereof.

Reverse Transcription Polymerase Chain Reaction (RT-PCR) can be utilizedin the present disclosure to determine expression of a gene to assistduring the selection step of a breeding scheme. It is a variant ofpolymerase chain reaction (PCR), a laboratory technique commonly used inmolecular biology to generate many copies of a DNA sequence, a processtermed “amplification”. In RT-PCR, however, RNA strand is first reversetranscribed into its DNA complement (complementary DNA, or cDNA) usingthe enzyme reverse transcriptase, and the resulting cDNA is amplifiedusing traditional or real-time PCR.

RT-PCR utilizes a pair of primers, which are complementary to a definedsequence on each of the two strands of the mRNA. These primers are thenextended by a DNA polymerase and a copy of the strand is made after eachcycle, leading to logarithmic amplification.

RT-PCR includes three major steps. The first step is the reversetranscription (RT) where RNA is reverse transcribed to cDNA using areverse transcriptase and primers. This step is very important in orderto allow the performance of PCR since DNA polymerase can act only on DNAtemplates. The RT step can be performed either in the same tube with PCR(one-step PCR) or in a separate one (two-step PCR) using a temperaturebetween 40° C. and 50° C., depending on the properties of the reversetranscriptase used.

The next step involves the denaturation of the dsDNA at 95° C., so thatthe two strands separate and the primers can bind again at lowertemperatures and begin a new chain reaction. Then, the temperature isdecreased until it reaches the annealing temperature which can varydepending on the set of primers used, their concentration, the probe andits concentration (if used), and the cation concentration. The mainconsideration, of course, when choosing the optimal annealingtemperature is the melting temperature (Tm) of the primers and probes(if used). The annealing temperature chosen for a PCR depends directlyon length and composition of the primers. This is the result of thedifference of hydrogen bonds between A-T (2 bonds) and G-C (3 bonds). Anannealing temperature about 5 degrees below the lowest Tm of the pair ofprimers is usually used.

The final step of PCR amplification is the DNA extension from theprimers which is done by the thermostable Taq DNA polymerase usually at72° C., which is the optimal temperature for the polymerase to work. Thelength of the incubation at each temperature, the temperaturealterations and the number of cycles are controlled by a programmablethermal cycler. The analysis of the PCR products depends on the type ofPCR applied. If a conventional PCR is used, the PCR product is detectedusing for example agarose gel electrophoresis or other polymer gel likepolyacrylamide gels and ethidium bromide (or other nucleic acidstaining).

Conventional RT-PCR is a time-consuming technique with importantlimitations when compared to real time PCR techniques. Furthermore, thespecificity of the assay is mainly determined by the primers, which cangive false-positive results. However, the most important issueconcerning conventional RT-PCR is the fact that it is a semi or even alow quantitative technique, where the amplicon can be visualized onlyafter the amplification ends.

Real time RT-PCR provides a method where the amplicons can be visualizedas the amplification progresses using a fluorescent reporter molecule.There are three major kinds of fluorescent reporters used in real timeRT-PCR, general nonspecific DNA Binding Dyes such as SYBR Green I,TaqMan Probes and Molecular Beacons (including Scorpions).

The real time PCR thermal cycler has a fluorescence detection threshold,below which it cannot discriminate the difference between amplificationgenerated signal and background noise. On the other hand, thefluorescence increases as the amplification progresses and theinstrument performs data acquisition during the annealing step of eachcycle. The number of amplicons will reach the detection baseline after aspecific cycle, which depends on the initial concentration of the targetDNA sequence. The cycle at which the instrument can discriminate theamplification generated fluorescence from the background noise is calledthe threshold cycle (Ct). The higher is the initial DNA concentration,the lower its Ct will be.

Other forms of nucleic acid detection can include next generationsequencing methods such as DNA SEQ or RNA SEQ using any known sequencingplatform including, but not limited to: Roche 454, Solexa GenomeAnalyzer, AB SOLiD, Illumina GA/HiSeq, Ion PGM, Mi Seq, among others(Liu et al, 2012 Journal of Biomedicine and Biotechnology Volume 2012 ID251364; Franca et al., 2002 Quarterly Reviews of Biophysics 35 pg.169-200; Mardis 2008 Genomics and Human Genetics vol. 9 pg. 387-402).

In other embodiments, nucleic acids may be detected with other highthroughput hybridization technologies including microarrays, gene chips,LNA probes, nanoStrings, and fluorescence polarization detection amongothers.

In some embodiments, detection of markers can be achieved at an earlystage of plant growth by harvesting a small tissue sample (e.g., branch,or leaf disk). This approach is preferable when working with largepopulations as it allows breeders to weed out undesirable progeny at anearly stage and conserve growth space and resources for progeny whichshow more promise. In some embodiments the detection of markers isautomated, such that the detection and storage of marker data is handledby a machine. Recent advances in robotics have also led to full serviceanalysis tools capable of handling nucleic acid/protein markerextractions, detection, storage and analysis.

Quantitative Trait Loci

Breeding schemes of the present application can include crosses betweendonor and recipient plants. In some embodiments said donor plantscontain a gene or genes of interest which may confer the plant with adesirable phenotype. The recipient line can be an elite line havingcertain favorable traits for commercial production. In one embodiment,the elite line may contain other genes that also impart said line withthe desired phenotype. When crossed together, the donor and recipientplant may create a progeny plant with combined desirable loci which mayprovide quantitatively additive effect of a particular characteristic.In that case, QTL mapping can be involved to facilitate the breedingprocess.

A QTL (quantitative trait locus) mapping can be applied to determine theparts of the donor plant's genome conferring the desirable phenotype,and facilitate the breeding methods. Inheritance of quantitative traitsor polygenic inheritance refers to the inheritance of a phenotypiccharacteristic that varies in degree and can be attributed to theinteractions between two or more genes and their environment. Though notnecessarily genes themselves, quantitative trait loci (QTLs) arestretches of DNA that are closely linked to the genes that underlie thetrait in question. QTLs can be molecularly identified to help mapregions of the genome that contain genes involved in specifying aquantitative trait. This can be an early step in identifying andsequencing these genes.

Typically, QTLs underlie continuous traits (those traits that varycontinuously, e.g. yield, height, level of resistance to virus, etc.) asopposed to discrete traits (traits that have two or several charactervalues, e.g. smooth vs. wrinkled peas used by Mendel in hisexperiments). Moreover, a single phenotypic trait is usually determinedby many genes. Consequently, many QTLs are associated with a singletrait.

A quantitative trait locus (QTL) is a region of DNA that is associatedwith a particular phenotypic trait. Knowing the number of QTLs thatexplains variation in the phenotypic trait tells about the geneticarchitecture of a trait. It may tell that a trait is controlled by manygenes of small effect, or by a few genes of large effect or by a severalgenes of small effect and few genes of larger effect.

Another use of QTLs is to identify candidate genes underlying a trait.Once a region of DNA is identified as contributing to a phenotype, itcan be sequenced. The DNA sequence of any genes in this region can thenbe compared to a database of DNA for genes whose function is alreadyknown.

In a recent development, classical QTL analyses are combined with geneexpression profiling i.e. by DNA microarrays. Such expression QTLs(e-QTLs) describes cis- and trans-controlling elements for theexpression of often disease-associated genes. Observed epistatic effectshave been found beneficial to identify the gene responsible by across-validation of genes within the interacting loci with metabolicpathway- and scientific literature databases.

QTL mapping is the statistical study of the alleles that occur in alocus and the phenotypes (physical forms or traits) that they produce(see, Meksem and Kahl, The handbook of plant genome mapping: genetic andphysical mapping, 2005, Wiley-VCH, ISBN 3527311165, 9783527311163).Because most traits of interest are governed by more than one gene,defining and studying the entire locus of genes related to a trait giveshope of understanding what effect the genotype of an individual mighthave in the real world.

Statistical analysis is required to demonstrate that different genesinteract with one another and to determine whether they produce asignificant effect on the phenotype. QTLs identify a particular regionof the genome as containing one or several genes, i.e. a cluster ofgenes that is associated with the trait being assayed or measured. Theyare shown as intervals across a chromosome, where the probability ofassociation is plotted for each marker used in the mapping experiment.

To begin, a set of genetic markers must be developed for the species inquestion. A marker is an identifiable region of variable DNA. Biologistsare interested in understanding the genetic basis of phenotypes(physical traits). The aim is to find a marker that is significantlymore likely to co-occur with the trait than expected by chance, that is,a marker that has a statistical association with the trait. Ideally,they would be able to find the specific gene or genes in question, butthis is a long and difficult undertaking. Instead, they can more readilyfind regions of DNA that are very close to the genes in question. When aQTL is found, it is often not the actual gene underlying the phenotypictrait, but rather a region of DNA that is closely linked with the gene.

For organisms whose genomes are known, one might now try to excludegenes in the identified region whose function is known with somecertainty not to be connected with the trait in question. If the genomeis not available, it may be an option to sequence the identified regionand determine the putative functions of genes by their similarity togenes with known function, usually in other genomes. This can be doneusing BLAST, an online tool that allows users to enter a primarysequence and search for similar sequences within the BLAST database ofgenes from various organisms.

Another interest of statistical geneticists using QTL mapping is todetermine the complexity of the genetic architecture underlying aphenotypic trait. For example, they may be interested in knowing whethera phenotype is shaped by many independent loci, or by a few loci, andhow do those loci interact. This can provide information on how thephenotype may be evolving.

Molecular markers are used for the visualization of differences innucleic acid sequences. This visualization is possible due to DNA-DNAhybridization techniques (RFLP) and/or due to techniques using thepolymerase chain reaction (e.g. STS, SNPs, microsatellites, AFLP). Alldifferences between two parental genotypes will segregate in a mappingpopulation based on the cross of these parental genotypes. Thesegregation of the different markers may be compared and recombinationfrequencies can be calculated. The recombination frequencies ofmolecular markers on different chromosomes are generally 50%. Betweenmolecular markers located on the same chromosome the recombinationfrequency depends on the distance between the markers. A lowrecombination frequency usually corresponds to a low distance betweenmarkers on a chromosome. Comparing all recombination frequencies willresult in the most logical order of the molecular markers on thechromosomes. This most logical order can be depicted in a linkage map(Paterson, 1996, Genome Mapping in Plants. R.G. Landes, Austin.). Agroup of adjacent or contiguous markers on the linkage map that isassociated to a reduced disease incidence and/or a reduced lesion growthrate pinpoints the position of a QTL.

The nucleic acid sequence of a QTL may be determined by methods known tothe skilled person. For instance, a nucleic acid sequence comprisingsaid QTL or a resistance-conferring part thereof may be isolated from adonor plant by fragmenting the genome of said plant and selecting thosefragments harboring one or more markers indicative of said QTL.Subsequently, or alternatively, the marker sequences (or parts thereof)indicative of said QTL may be used as (PCR) amplification primers, inorder to amplify a nucleic acid sequence comprising said QTL from agenomic nucleic acid sample or a genome fragment obtained from saidplant. The amplified sequence may then be purified in order to obtainthe isolated QTL. The nucleotide sequence of the QTL, and/or of anyadditional markers comprised therein, may then be obtained by standardsequencing methods.

One or more such QTLs associated with a desirable trait in a donor plantcan be transferred to a recipient plant to incorporate the desirabletrait into progeny plants by transferring and/or breeding methods.

In one embodiment, an advanced backcross QTL analysis (AB-QTL) is usedto discover the nucleotide sequence or the QTLs responsible for theresistance of a plant. Such method was proposed by Tanksley and Nelsonin 1996 (Tanksley and Nelson, 1996, Advanced backcross QTL analysis: amethod for simultaneous discovery and transfer of valuable QTL fromun-adapted germplasm into elite breeding lines. Theor Appl Genet92:191-203) as a new breeding method that integrates the process of QTLdiscovery with variety development, by simultaneously identifying andtransferring useful QTL alleles from un-adapted (e.g., land races, wildspecies) to elite germplasm, thus broadening the genetic diversityavailable for breeding. AB-QTL strategy was initially developed andtested in tomato, and has been adapted for use in other crops includingrice, maize, wheat, pepper, barley, and bean. Once favorable QTL allelesare detected, only a few additional marker-assisted generations arerequired to generate near isogenic lines (NILs) or introgression lines(ILs) that can be field tested in order to confirm the QTL effect andsubsequently used for variety development.

Isogenic lines in which favorable QTL alleles have been fixed can begenerated by systematic backcrossing and introgressing of marker-defineddonor segments in the recurrent parent background. These isogenic linesare referred to as near isogenic lines (NILs), introgression lines(ILs), backcross inbred lines (BILs), backcross recombinant inbred lines(BCRIL), recombinant chromosome substitution lines (RCSLs), chromosomesegment substitution lines (CSSLs), and stepped aligned inbredrecombinant strains (STAIRSs). An introgression line in plant molecularbiology is a line of a crop species that contains genetic materialderived from a similar species. ILs represent NILs with relatively largeaverage introgression length, while BILs and BCRILs are backcrosspopulations generally containing multiple donor introgressions per line.As used herein, the term “introgression lines or ILs” refers to plantlines containing a single marker defined homozygous donor segment, andthe term “pre-ILs” refers to lines which still contain multiplehomozygous and/or heterozygous donor segments.

To enhance the rate of progress of introgression breeding, a geneticinfrastructure of exotic libraries can be developed. Such an exoticlibrary comprises a set of introgression lines, each of which has asingle, possibly homozygous, marker-defined chromosomal segment thatoriginates from a donor exotic parent, in an otherwise homogenous elitegenetic background, so that the entire donor genome would be representedin a set of introgression lines. A collection of such introgressionlines is referred as libraries of introgression lines or IL libraries(ILLs). The lines of an ILL cover usually the complete genome of thedonor, or the part of interest. Introgression lines allow the study ofquantitative trait loci, but also the creation of new varieties byintroducing exotic traits. High resolution mapping of QTL using ILLsenable breeders to assess whether the effect on the phenotype is due toa single QTL or to several tightly linked QTL affecting the same trait.In addition, sub-ILs can be developed to discover molecular markerswhich are more tightly linked to the QTL of interest, which can be usedfor marker-assisted breeding (MAB). Multiple introgression lines can bedeveloped when the introgression of a single QTL is not sufficient toresult in a substantial improvement in agriculturally important traits(Gur and Zamir, Unused natural variation can lift yield barriers inplant breeding, 2004, PLoS Biol.; 2(10):e245).

Tissue Culture

As it is well known in the art, tissue culture of tomato can be used forthe in vitro regeneration of tomato plants. Tissues cultures of varioustissues of tomato and regeneration of plants therefrom are well knownand published. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants as described in Girish-Chandelet al., Advances in Plant Sciences. 2000, 13: 1, 11-17, Costa et al.,Plant Cell Report. 2000, 19: 3327-332, Plastira et al., ActaHorticulturae. 1997, 447, 231-234, Zagorska et al., Plant Cell Report.1998, 17: 12 968-973, Asahura et al., Breeding Science. 1995, 45:455-459, Chen et al., Breeding Science. 1994, 44: 3, 257-262, Patil etal., Plant and Tissue and Organ Culture. 1994, 36: 2, 255-258. It isclear from the literature that the state of the art is such that thesemethods of obtaining plants are routinely used and have a very high rateof success. Thus, another aspect of this invention is to provide cellswhich upon growth and differentiation produce tomato plants having thephysiological and morphological characteristics of hybrid tomato plantHM5235.

As used herein, the term “tissue culture” indicates a compositioncomprising isolated cells of the same or a different type or acollection of such cells organized into parts of a plant. Exemplarytypes of tissue cultures are protoplasts, calli, plant clumps, and plantcells that can generate tissue culture that are intact in plants orparts of plants, such as embryos, pollen, flowers, seeds, leaves, stems,roots, root tips, anthers, pistils, meristematic cells, axillary buds,ovaries, seed coat, endosperm, hypocotyls, cotyledons and the like.Means for preparing and maintaining plant tissue culture are well knownin the art. By way of example, a tissue culture comprising organs hasbeen used to produce regenerated plants. U.S. Pat. Nos. 5,959,185,5,973,234, and 5,977,445 describe certain techniques, the disclosures ofwhich are incorporated herein by reference.

EXAMPLES

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

Example 1—Development of New 11M5235 Tomato Variety

Hybrid tomato plant HM5235 has superior characteristics. The femalePTOMF6 and male PTOMM6 parents were crossed to produce hybrid (F1) seedsof HM5235. The seeds of HM5235 can be grown to produce hybrid plants andparts thereof. The hybrid HM5235 can be propagated by seeds by crossingtomato inbred line PTOMF6 with tomato inbred line PTOMM6 orvegetatively.

Breeding History—The origin and breeding history of hybrid plant HM5235can be summarized as follows: the line PTOMF6 was used as the femaleplant and crossed by pollen from the line PTOMM6 (both proprietary linesowned by HM.CLAUSE, Inc.). The first trial planting of this hybrid wasdone in four different locations (from Fresno County to Yolo County) inCalifornia in the summer of the first year of development. The hybridwas further trialed for two additional years, an example of such trialbeing disclosed in Tables 2 and 3.

The inbred line PTOMF6 is a parent with strong vine, produces small tomedium size firm fruits, this inbred line was used as female parent inthis cross.

The inbred PTOMM6 is a large plant with large firm red fruits, it wasused as the male parent in this cross.

Hybrid tomato plant HM5235 is similar to hybrid tomato plant BQ141.BQ141 is a commercial variety. As shown in Tables 2 and 3, while similarto hybrid tomato plant BQ141, there are significant differencesincluding the brix value of fruits extract which is 5.73 for HM5235while it is 4.98 for BQ141, the average fruit yield for five plantswhich is 88.13 pounds for HM5235 while it is 75.21 for BQ141, the fruitOstwald value which is 361.41 for HM5235 and 450.82 for BQ141.

Some of the criteria used to select the hybrid HM5235 as well as theirinbred parent lines in various generations include: earliness, yield,brix, viscosity (paste and serum thickness), fruit weight, fruitfirmness, fruit color and disease resistance.

TABLE 1 Comparison between HM5235 and BQ141 HM5235 BQ141 Observationtrial planted in: Field Field Observation trial planting type:Transplant Transplant Dates of seeding/transplanting: March to May Marchto May Location of trials: Fresno, Kern, King, Yolo, Fresno, Kern, King,Yolo, Stockton, Stockton, Sacramento, California Sacramento, CaliforniaObservation trial planting type: Transplanted and unstaked Transplantedand unstaked Seedling anthocyanin in hypocotyl of 2-15 cm: 2 2 1 =absent; 2 = present Mature plant: height 55.1 cm  53.0 cm  growth type:1 = 2 2 indeterminate; 2 = determinate Plant form: 1 = normal; 2 = 1 2compact; 3 = dwarf; 4 = brachytic size of canopy (compared to 3 2 othersof similar form); 1 = small; 2 = medium; 3 = large; habit: 1 =sprawling; 2 = semi- 2 2 erect; 3 = erect branching: 1 = sparse; 2 = 2 2intermediate; 3 = profuse number of nodes between 1 to 2 1 to 2inflorescence pubescence on younger stems: 1 = 2 2 smooth (no longhairs); 2 = sparsely hairy (scattered long hairs); 3 = moderately hairy;4 = densely hairy or wooly Leaf type: 1 = tomato; 2 = potato 1 1(Trip-L-Crop) Morphology margins of major leaflets: 1 = 2 2 absent; 2 =shallowly toothed or scalloped; 3 = deeply toothed or cut, speciallytowards base surface of major leaflets: 1 = 1 1 smooth; 2 = rogues(bumpy or veiny) pubescence: 1 = smooth (no long 2 2 hairs); 2 = normal;3 = hirsute; 4 = wooly Inflorescence Type: 1 = simple; 2 = forked (2 1 +2 1 + 2 major axes); 3 = compound (much branched) number of flowers in 76 inflorescence average leafy or “running” 1 1 inflorescence: 1 =absent; 2 = occasional; 3 = frequent Flower calyx: 1 = normal, lobesawl- 1 1 shaped; 2 = macrocalyx, lobes large, 3 = fleshy calyx-lobes: 1= shorter than 2 1 corolla; 2 = approx., equaling corolla; 3 =distinctly longer than corolla corolla color: 1 = yellow: 2 = old 1 1gold; 3 = white or tan style pubescence: 1 = absent; 2 = 1 1 sparse; 3 =dense anthers: 1 = all fused into tube; 2 = 1 1 separating into 2 ormore Fruit typical shape in longitudinal date-like date-like sectionshape of transverse section: 1 = 2 2 round; 2 = flattened; 3 = angular;4 = irregular shape of stem end: 1 = flat; 2 = 2 2 indented shape ofblossom end: 1 = 2 2 indented; 2 = flat; 3 = nippled; 4 = tapered shapeof pistil scar: 1 = dot; 2 = 1 1 stellate; 3 = linear; 4 = irregularabscission layer: 1 = present 2 2 (pedicellate); 2 = absent (jointless)Length of mature fruit (stem 63.1 mm 59.7 mm axis) Diameter of fruit atwidest point 44.8 mm 44.3 mm Number of locules: 1 = two; 2 = 1 + 2 1 + 2three; 3 = four or five; 4 = more than 5 Fruit base color (mature-green3 3 stage): 1 = light green; 2 = light gray-green; 3 = apple or mediumgreen 4 = yellow green; 5 = dark green Fruit pattern (mature-green 1 1stage): 1 = uniform green; 2 = green-shouldered; 3 = radial stripes onsides of fruit Fruit color full ripe: 1 = white; 2 = 5 5 yellow; 3 =orange; 4 = pink; 5 = red; 6 = brownish; 7 = greenish; 8 = other Fleshcolor full ripe: 1 = 3 3 yellow; 2 = pink; 3 = red/crimson; 4 = orange;5 other Flesh color: 1 = uniform; 2 = 1 1 with lighter and darker areasin walls locular gel color of table-ripe 3 3 fruit: 1 = green; 2 =yellow; 3 = red ripening: 1 = inside out; 2 = 1 1 uniformity; 3 =outside in stem scar size: 1 = small 2 2 (Roma); 2 = medium; 3 = largecore: 1 = coreless (absent or 2 2 smaller than 6 × 6 mm); 2 = presentepidermis color: 1 = colorless; 2 = 2 2 yellow epidermis: 1 = normal; 2= easy- 2 2 peel Field holding ability no no Fruit harvestability: 1 =many 4 4 rotten or broken; 2 = fruit soft, many rotten fruits; 3 = somerotten fruit; 4 = few rotten fruit; 5 = no rotten fruits, no rejectedfruits Disease and pest reaction: 0 = not tested; 1 = highly resistant;2 = resistant, few symptoms; 3 = resistance, few lesions in number andsize; 4 = moderately resistance; 5 = intermediate resistance; 6 =moderate susceptible; 7 = susceptible; 9 = highly susceptible Virusdiseases curly top 0 0 tobacco mosaic race 0 7 7 tobacco mosaic race 1 77 tobacco mosaic race 2 7 7 tobacco mosaic race 2² 7 7 Tomato spottedwilt 2 2 Tomato yellow leaf curl 7 7 Others 0 0 Bacterial diseaseBacterial canker 0 0 (Corynebacterium michiganense) Bacterial speck(Pseudomonas 7 7 tomato) race 0 Bacterial spot (Xanthomonas 0 0vesicatorium) Other bacterial disease 0 0 Fungal diseases Fusarium wiltrace 1 (F. oxysporum 2 2 f. lycopersici) Fusarium wilt race 2 (F.oxysporum 2 2 f. lycopersici) Fusarium wilt race 3 (F. oxysporum 2 2 f.lycopersici) Late blight, race 0 0 0 (Phytophthora infestans) Lateblight, race 1 0 0 Verticillium wilt race 1 (V. albo- 2 2 atrum)Verticillium wilt race 2 0 0 Other fungal disease 0 0 Insects and Pestssouthern root knot nematode 2 2 (M. incognia) whitefly (T. vaporariorum)0 0 Other 0 0 Seedling (Maturity in number of 122 days 122 days days) toonce harvest Fruit season (concentration): 1 = 4 4 long (Marglobe); 2 =medium (Westover); 3 = short, concentrated; 4 = very concentrated (UC82)Relative maturity in areas 4 4 tested: 1 = early; 2 = medium early; 3 =medium; 4 = medium late; 5 = late; 6 = variable Adaptation Culture: 1 =field; 2 = 1 1 greenhouse Principle use(s): 1 = home 3, 4, 5 3, 4, 5garden; 2 = fresh market; 3 = whole-pack canning; 4 = concentratedproducts; 5 = multiuse; 6 = other Average yield (ton/acre) in  66.25 56.74 California (grower's field, 20 data points) Machine harvest: 1 =not 2 2 adapted; 2 = adapted Regions to which adaptation has 9 + 11 + 129 + 11 + 12 been demonstrated: 1 = Northeast; 2 = Mid Atlantic; 3 =Southeast; 4 = Florida; 5 = Great Plains; 6 = south central; 7 =Intermountain West; 8 = Northwest; 9 = California (Sacramento and UpperSan Joaquin Valley); 10 = California (Coastal Areas); 11 = California(Southern San Joaquin Valley & desserts); 12 = South American countries

The hybrid tomato plant HM5235 has shown uniformity and stability forthe traits, within the limits of environmental influence for the traitsas described in the following Variety Descriptive Information. Novariant traits have been observed or are expected for agronomicalimportant traits in tomato hybrid HM5235.

Example 2—Comparison of New HM5235 Tomato with Check Variety

In the tables that follow, the traits and characteristics of hybridtomato HM5235 are given compared to another hybrid. The data collectedare presented for key characteristics and traits. Hybrid tomato HM5235was tested at numerous locations, with two or more replications perlocation. Information about the hybrid, as compared to several checkhybrid is presented (based primarily on data collected in California,all experiments done under the direct supervision of the applicant).

Table 2 below shows the characteristics of hybrid tomato HM5235 comparedto hybrid BQ141 as measured in California from July to September. Column1 identifies the varieties, column 2 described disease resistancepackage (V is Verticillium resistance, F is Fusarium Race 1 resistance,F2 is Fusarium Race 2 resistance, F3 is Fusarium Race 3 resistance, N isNematode resistance, Sw is Tomato Spotted Wilt Virus Resistance), column3 the average of fruit puree acidity measured by pH, column 4 the fruitraw brix or soluble solid content, column 5 the average of JuiceBostwick (JB), column 6 the serum viscosity (Ostwald).

TABLE 2 Disease Variety Resistance pH Brix Bostwick Ostwald HM5235VFF2F3NSw 4.53 5.73 13.71 361.41 BQ141 VFF2F3NSw 4.51 4.98 15.21 450.82

Table 3 below shows the characteristics of hybrid tomato HM5235 comparedto hybrid BQ141 as measured in California from July to September. Column1 identifies the varieties, column 2 the average single fruit weight(grams), column 3 the average fruit firmness as measured by “P5” scale,column 4 the average fruit color as measured by ration of a/b ratio offruit puree, column 5 the average fruit yield of 5 plants (in pounds).

TABLE 3 Variety Frt. Wt. (g) Firmness (P5) Color YLD AVE HM5235 82.964.62 2.29 88.13 BQ141 80.09 4.37 2.24 75.21

DEPOSIT INFORMATION

A deposit of the tomato seed of this invention is maintained byHM.CLAUSE, Inc. Davis Research Station, 9241 Mace Boulevard, Davis,Calif. 95616. In addition, a sample of the hybrid tomato seed of thisinvention has been deposited with the National Collections ofIndustrial, Food and Marine Bacteria (NCIMB), 23 St Machar Drive,Aberdeen, Scotland, AB24 3RY, United Kingdom.

To satisfy the enablement requirements of 35 U.S.C. 112, and to certifythat the deposit of the isolated strain of the present invention meetsthe criteria set forth in 37 CFR 1.801-1.809, Applicants hereby make thefollowing statements regarding the deposited hybrid tomato HM5235(deposited as NCIMB Accession No. 42747).

1. During the pendency of this application, access to the invention willbe afforded to the Commissioner upon request;

2. All restrictions on availability to the public will be irrevocablyremoved upon granting of the patent under conditions specified in 37 CFR1.808;

3. The deposit will be maintained in a public repository for a period of30 years or 5 years after the last request or for the effective life ofthe patent, whichever is longer;

4. A test of the viability of the biological material at the time ofdeposit will be conducted by the public depository under 37 CFR 1.807;and

5. The deposit will be replaced if it should ever become unavailable.

Access to this deposit will be available during the pendency of thisapplication to persons determined by the Commissioner of Patents andTrademarks to be entitled thereto under 37 C.F.R. § 1.14 and 35 U.S.C. §122. Upon allowance of any claims in this application, all restrictionson the availability to the public of the variety will be irrevocablyremoved by affording access to a deposit of at least 2,500 seeds of thesame variety with the NCIMB.

INCORPORATION BY REFERENCE

All references, articles, publications, patents, patent publications,and patent applications cited herein are incorporated by reference intheir entireties for all purposes.

However, mention of any reference, article, publication, patent, patentpublication, and patent application cited herein is not, and should notbe taken as an acknowledgment or any form of suggestion that theyconstitute valid prior art or form part of the common general knowledgein any country in the world.

The invention claimed is:
 1. A seed of hybrid tomato designated HM5235,wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 42747. 2. A tomato plant, a plant partthereof, or a plant cell thereof, produced by growing the seed of claim1, wherein a plant regenerated from the plant part or the plant cell hasall of the physiological and morphological characteristics of hybridtomato HM5235 listed in Table 1 when grown under the same environmentalconditions.
 3. The tomato part of claim 2, wherein the tomato part isselected from the group consisting of a leaf, a flower, a fruit, a cell,a rootstock, and a scion.
 4. A tomato plant, a plant part, or a plantcell thereof, wherein the plant, a plant regenerated from the plant partor the plant cell has all of the physiological and morphologicalcharacteristics of hybrid HM5235 listed in Table 1 when grown under thesame environmental conditions, wherein a representative sample of seedof hybrid HM5235 has been deposited under NCIMB No.
 42747. 5. A tomatoplant, a plant part, or a plant cell thereof, wherein the plant, a plantregenerated from the plant part or the plant cell has all of thephysiological and morphological characteristics of hybrid HM5235,wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 42747. 6. A tissue culture of regenerablecells produced from the plant or plant part of claim 2, wherein a plantregenerated from the tissue culture has all of the physiological andmorphological characteristics of hybrid HM5235 listed in Table 1 whengrown in the same environmental conditions.
 7. A tomato plantregenerated from the tissue culture of claim 6, said plant having allthe physiological and morphological characteristics of hybrid HM5235,listed in Table 1 when grown under the same environmental conditions,wherein a representative sample of seed of said hybrid has beendeposited under NCIMB No.
 42747. 8. A tomato fruit produced from theplant of claim
 2. 9. A method for harvesting a tomato fruit comprisinga) growing the tomato plant of claim 2 to produce a tomato fruit, and b)harvesting said tomato fruit.
 10. A tomato fruit produced by the methodof claim
 9. 11. A method for producing a tomato seed comprising crossinga first parent tomato plant with a second parent tomato plant andharvesting the resultant tomato seed, wherein said first parent tomatoplant and/or second parent tomato plant is the tomato plant of claim 2.12. A method for producing a tomato seed comprising self-pollinating thetomato plant of claim 2 and harvesting the resultant tomato seed.
 13. Amethod of vegetatively propagating the tomato plant of claim 2, saidmethod comprising a) collecting part of the plant of claim 2 and b)regenerating a plant from said part.
 14. The method of claim 13 furthercomprising harvesting a fruit from said plant.
 15. A plant obtained fromthe method of claim 13, wherein the plant has all of the physiologicaland morphological characteristics of hybrid HM5235 listed in Table 1when grown under the same environmental conditions.
 16. A fruit obtainedfrom the plant of claim
 15. 17. A method of producing a tomato plantderived from the hybrid variety HM5235, the method comprising: (a)self-pollinating the plant of claim 2 at least once to produce a progenyplant.
 18. The method of claim 17 further comprising the steps of: (b)crossing the progeny plant derived from the variety HM5235 with itselfor a second tomato plant to produce a seed of progeny plant ofsubsequent generation; (c) growing the progeny plant of the subsequentgeneration from the seed; and (d) crossing the progeny plant of thesubsequent generation with itself or a second tomato plant to produce atomato plant derived from the tomato hybrid variety HM5235.
 19. Themethod of claim 18 further comprising the step of (e) repeating step b)and step c) using tomato plant obtained in step (d) to produce a tomatoplant further derived from the variety HM5235.
 20. A method of producinga tomato plant derived from the hybrid variety HM5235, the methodcomprising: (a) crossing the plant of claim 2 with a second tomato plantto produce a progeny plant.
 21. The method of claim 20 furthercomprising the steps of: (b) crossing the progeny plant derived from thevariety HM5235 with itself or a second tomato plant to produce a seed ofprogeny plant of subsequent generation; (c) growing the progeny plant ofthe subsequent generation from the seed; and (d) crossing the progenyplant of the subsequent generation with itself or a second tomato plantto produce a tomato plant derived from the tomato hybrid variety HM5235.22. The method of claim 21 further comprising the step of (e) repeatingstep b) and step c) using tomato plant obtained in step (d) to produce atomato plant further derived from tomato hybrid HM5235.
 23. A tomatoplant further comprising a single locus conversion and otherwise all ofthe physiological and morphological characteristics of hybrid HM5235listed in Table 1 when grown under the same environmental conditions,wherein a representative sample of seed of said hybrid HM5235 has beendeposited under NCIMB No.
 42747. 24. The plant of claim 23 wherein thesingle locus conversion confers said plant with herbicide resistance.25. The plant of claim 23, wherein the single locus conversion is anartificially mutated gene or an artificially mutated nucleotidesequence.
 26. The plant of claim 23 wherein the single locus conversionis a gene that has been modified through the use of a breeding techniqueselected from the group consisting of Zinc finger nuclease (ZFN)technology, oligonucleotide directed mutagenesis, cisgenesis andintragenesis, RNA-dependent DNA methylation, reverse breeding,agro-infiltration, Transcription Activation-Like Effector Nuclease(TALENs), CRISPR/Cas system, engineered meganuclease re-engineeredhoming endonuclease, and DNA guided genome editing.
 27. A method ofproducing a tomato plant, comprising using tomato hybrid HM5235 as arootstock or a scion to produce a grafted tomato plant, wherein arepresentative sample of seed of said tomato hybrid HM5235 has beendeposited under NCIMB No.
 42747. 28. A method for producing nucleicacids, the method comprising isolating nucleic acids from the plant ofclaim
 2. 29. A method for producing a second tomato plant, the methodcomprising applying plant breeding techniques to the plant or plant partof claim 2 to produce the second tomato plant.